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US20040115675A1 - Regulation of human transient receptor potential channel - Google Patents

Regulation of human transient receptor potential channel Download PDF

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US20040115675A1
US20040115675A1 US10/467,163 US46716303A US2004115675A1 US 20040115675 A1 US20040115675 A1 US 20040115675A1 US 46716303 A US46716303 A US 46716303A US 2004115675 A1 US2004115675 A1 US 2004115675A1
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transient receptor
receptor potential
polypeptide
potential channel
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Timothy Smith
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Bayer AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

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  • the invention relates to the area of ion channel regulation.
  • Ion channels are integral membrane proteins, typically comprising multiple subunits, which form selective and highly regulated pores in cellular membranes. Each of these pores controls the influx and efflux of a given ion (e.g., sodium, potassium, calcium, or chloride) across the plasma membrane or the membranes of intracellular compartmnents.
  • a given ion e.g., sodium, potassium, calcium, or chloride
  • Many important physiological processes depend on the control of ion gradients by ion channels. Such processes include synaptic transmission, secretion, fertilization, muscle contraction, and regulation of intracellular and extracellular ion concentrations and pH.
  • Ion channels open in response to various stimuli. For example, there are ligand-gated channels, second messenger-gated channels, voltage-gated channels, and shear- or stress-gated channels.
  • Certain channels allow ions to leak across membranes without a specific stimulus.
  • the gating properties characteristic of a given channel include the period of time it is open, the frequency of opening, the strength of stimulus required for activation, and the refractory period. These characteristics can vary depending on the subunit composition of the channel, association of the channel with accessory proteins, and phosphorylation or other post-translational modification of channel polypeptides. See, e.g., U.S. Pat. No. 6,071,720.
  • transient receptor potential channel polypeptide comprising an amino acid sequence selected from the group consisting of:
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 2;
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 11;
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 12;
  • Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a transient receptor potential channel polypeptide comprising an amino acid sequence selected from the group consisting of:
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 2;
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 11;
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 12;
  • binding between the test compound and the transient receptor potential channel poly-peptide is detected.
  • a test compound which binds to the transient receptor potential channel polypeptide is thereby identified as a potential agent for decreasing extra-cellular matrix degradation.
  • the agent can work by decreasing the activity of the transient receptor potential channel.
  • Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation.
  • a test compound is contacted with a polynucleotide encoding a transient receptor potential channel polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 9;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10;
  • a test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation.
  • the agent can work by decreasing the amount of the transient receptor potential channel through interacting with the transient receptor potential channel mRNA.
  • Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation.
  • a test compound is contacted with a transient receptor potential channel polypeptide comprising an amino acid sequence selected from the group consisting of:
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 2;
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 11;
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 12;
  • a transient receptor potential channel activity of the polypeptide is detected.
  • a test compound which increases transient receptor potential channel activity of the polypeptide relative to transient receptor potential channel activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation.
  • a test compound which decreases transient receptor potential channel activity of the polypeptide relative to transient receptor potential channel activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • a test compound is contacted with a transient receptor potential channel product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 9;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10;
  • Binding of the test compound to the transient receptor potential channel product is detected.
  • a test compound which binds to the transient receptor potential channel product is thereby identified as a potential agent for decreasing extracellular matrix degradation.
  • Still another embodiment of the invention is a method of reducing extracellular matrix degradation.
  • a cell is contacted with a reagent which specifically binds to a polynucleotide encoding a transient receptor potential channel polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 9;
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10;
  • Transient receptor potential channel activity in the cell is thereby decreased.
  • the invention thus provides a human transient receptor potential channel that can be used to identify test compounds that may act, for example, as activators or inhibitors of human transient receptor potential channel.
  • Human transient receptor potential channel and fragments thereof also are useful in raising specific antibodies that can block the polypeptide and effectively reduce its activity.
  • FIG. 1 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:1).
  • FIG. 2 shows the amino acid sequence deduced from the DNA-sequence of FIG. 1 (SEQ ID NO:2).
  • FIG. 3 shows the amino acid sequence of a protein identified by swissnew
  • FIG. 4 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:4).
  • FIG. 5 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:5).
  • FIG. 6 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:6).
  • FIG. 7 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:7).
  • FIG. 8 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO 8:).
  • FIG. 9 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:9).
  • FIG. 10 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:10).
  • FIG. 11 shows the amino acid sequence deduced from the DNA-sequence of FIG. 9 (SEQ ID NO:11).
  • FIG. 12 shows the amino acid sequence deduced from the DNA-sequence of FIG. 10 (SEQ ID NO:12).
  • FIG. 13 shows the BLASTP—alignment of 302_prot (SEQ ID NO:2) against swissnew
  • FIG. 14 shows the HMMPFAM—alignment of 302_prot (SEQ ID NO:2) against pfam
  • FIG. 15 shows the HMMPFAM—alignment of 302_from_mouse against pfam
  • FIG. 16 shows the genewise output
  • the invention relates to an isolated polynucleotide from the group consisting of:
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 2;
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 11;
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 12;
  • Human transient receptor potential channel comprises the amino acid sequence shown in SEQ ID NO:2.
  • a coding sequence for human transient receptor potential channel is shown in SEQ ID NO:1. This sequence is contained within the longer sequence shown in SEQ ID NO:4. This sequence is located on chromosome 9.
  • Related ESTs (SEQ ID NOS:5-8) are expressed in kidney and retina.
  • Pfamsearch identified a TRPC family domain (PF02164) in SEQ ID NO:2.
  • a multiple sequence alignment of SEQ ID NO:2 with other TRPCs revealed additional conserved regions.
  • hydrophobicity analysis predicted six membrane-spanning domains for the human transient receptor potential channel of the invention, which is a characteristic number for TRPCs.
  • Human transient receptor potential channel is 25% identical over 896 amino acids to swissnew
  • Human transient receptor potential channel of the invention is expected to be useful for the same purposes as previously identified transient receptor potential channels. Human transient receptor potential channel is believed to be useful in therapeutic methods to treat disorders such as cancer, cardiovascular disorders, and CNS disorders. Human transient receptor potential channel also can be used to screen for human transient receptor potential channel activators and inhibitors.
  • Human transient receptor potential channel polypeptides comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1133 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof, as defined below.
  • a transient receptor potential channel polypeptide of the invention therefore can be a portion of a transient receptor potential channel protein, a full-length transient receptor potential channel protein, or a fusion protein comprising all or a portion of a transient receptor potential channel protein.
  • transient receptor potential channel polypeptide variants that are biologically active, e.g., retain the ability to function as an ion channel, also are transient receptor potential channel polypeptides.
  • naturally or non-naturally occurring transient receptor potential channel polypeptide variants have amino acid sequences which are at least about 26, 30, 35, 40, 45, 50, 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, 96, 98, or 99% identical to the amino acid sequence shown in SEQ ID NO:2 or a fragnent thereof. Percent identity between a putative transient receptor potential channel polypeptide variant and an amino acid sequence of SEQ ID NO:2 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes).
  • Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions.
  • Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a transient receptor potential channel polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active transient receptor potential channel polypeptide can readily be determined by assaying for functional activity, as described for example, in the “Functional Assays” section, below.
  • Fusion proteins are useful for generating antibodies against transient receptor potential channel polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a transient receptor potential channel polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.
  • a transient receptor potential channel polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1133 contiguous amino acids of SEQ ID NO:2 or of a biologically active variant, such as those described above.
  • the first polypeptide segment also can comprise full-length transient receptor potential channel protein.
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include ⁇ -galactosidase, ⁇ -glucuonidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.
  • a fusion protein also can be engineered to contain a cleavage site located between the transient receptor potential channel polypeptide-encoding sequence and the heterologous protein sequence, so that the transient receptor potential channel polypeptide can be cleaved and purified away from the heterologous moiety.
  • a fusion protein can be synthesized chemically, as is known in the art.
  • a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology.
  • Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO:1 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art.
  • kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountan View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).
  • Species homologs of human transient receptor potential channel polypeptide can be obtained using transient receptor potential channel polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifyig cDNAs which encode homologs of transient receptor potential channel polypeptide, and expressing the cDNAs as is known in the art.
  • a transient receptor potential channel polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a transient receptor potential channel polypeptide.
  • a coding sequence for human transient receptor potential channel is shown in SEQ ID NO:1.
  • nucleotide sequences encoding human transient receptor potential channel polypeptides as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in SEQ ID NO:1 or its complement also are transient receptor potential channel polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of ⁇ 12 and a gap extension penalty of ⁇ 2.
  • transient receptor potential channel polynucleotides that encode biologically active transient receptor potential channel polypeptides also are transient receptor potential channel polynucleotides.
  • Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO:1 or its complement also are transient receptor potential channel polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.
  • transient receptor potential channel polynucleotides variants and homologs of the transient receptor potential channel polynucleotides described above also are transient receptor potential channel polynucleotides.
  • homologous transient receptor potential channel polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known transient receptor potential channel polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions—2 ⁇ SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2 ⁇ SSC, 0.1% SDS, 50° C.
  • homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.
  • Species homologs of the transient receptor potential channel polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of transient receptor potential channel polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T m of a double-stranded DNA decreases by 1-1.5° C. with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973).
  • Variants of human transient receptor potential channel polynucleotides or transient receptor potential channel polynucleotides of other species can therefore be identified by hybridizing a putative homologous transient receptor potential channel polynucleo-tide with a polynucleotide having a nucleotide sequence of SEQ ID NO:1 or the complement thereof to form a test hybrid.
  • the melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
  • Nucleotide sequences which hybridize to transient receptor potential channel poly-nucleotides or their complements following stringent hybridization and/or wash conditions also are transient receptor potential channel polynucleotides.
  • Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.
  • T m a combination of temperature and salt concentration should be chosen that is approximately 12-20° C. below the calculated T m of the hybrid under study.
  • the T m of a hybrid between a transient receptor potential channel polynucleotide having a nucleotide sequence shown in SEQ ID NO:1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
  • T m 81.5° C. ⁇ 16.6(log 10 [Na + ])+0.41(%G+C) ⁇ 0.63(%formamide) ⁇ 600/ l ),
  • Stringent wash conditions include, for example, 4 ⁇ SSC at 65° C., or 50% formamide, 4 ⁇ SSC at 42° C., or 0.5 ⁇ SSC, 0.1% SDS at 65° C.
  • Highly stringent wash conditions include, for example, 0.2 ⁇ SSC at 65° C.
  • a transient receptor potential channel polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Poly-nucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated transient receptor potential channel polynucleotides.
  • restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise transient receptor potential channel nucleotide sequences.
  • Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.
  • Human transient receptor potential channel cDNA molecules can be made with standard molecular biology techniques, using transient receptor potential channel mRNA as a template. Human transient receptor potential channel cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • transient receptor potential channel polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof.
  • the partial sequence disclosed herein can be used to identify the corresponding full length gene from which it was derived.
  • the partial sequence can be nick-translated or end-labeled with 32 P using polynucleotide kinase using labeling methods known to those with skill in the art (BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al., eds., Elsevier Press, N.Y., 1986).
  • a lambda library prepared from human tissue can be directly screened with the labeled sequences of interest or the library can be converted en masse to pBluescript (Stratagene Cloning Systems, La Jolla, Calif. 92037) to facilitate bacterial colony screening (see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press (1989, pg. 1.20).
  • Positive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial sequence and the other primer from the vector.
  • Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysis.
  • the complete sequence of the clones can be determined, for example after exonuclease III digestion (McCombie et al., Methods 3, 33-40, 1991). A series of deletion clones are generated, each of which is sequenced. The resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence.
  • PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al., Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72° C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic. 1, 111-119, 1991).
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5′ regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5′ non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.
  • Human transient receptor potential channel polypeptides can be obtained, for example, by purification from human cells, by expression of transient receptor potential channel polynucleotides, or by direct chemical synthesis.
  • Human transient receptor potential channel polypeptides can be purified from any cell that expresses the polypeptide, including host cells that have been transfected with transient receptor potential channel expression constructs.
  • a purified transient receptor potential channel polypeptide is separated from other compounds that normally associate with the transient receptor potential channel polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • a preparation of purified transient receptor potential channel polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.
  • the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding transient receptor potential channel polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding a transient receptor potential channel polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus,
  • control elements or regulatory sequences are those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagernid (Stratagene, LaJolla, Calif.) or pSPORT1 plasmid (Life Technologies) and the like can be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagernid (Stratagene, LaJolla, Calif.) or pSPORT1 plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a transient receptor potential channel polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected depending upon the use intended for the transient receptor potential channel polypeptide. For example, when a large quantity of a transient receptor potential channel polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding the transient receptor potential channel polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • BLUESCRIPT a sequence encoding the transient receptor potential channel polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced.
  • pIN vectors Van Heeke & Schuster, J BioL Chem. 264, 5503-5509, 1989
  • pGEX vectors Promega, Madison, Wis.
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • transient receptor potential channel polypeptides can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al., EMBO J. 3, 1671-1680, 1984; Broglie et al., Science 224, 838-843, 1984; Winter et al., Results Probl. Cell Differ. 17, 85-105, 1991).
  • constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
  • pathogen-mediated transfection Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).
  • An insect system also can be used to express a transient receptor potential channel polypeptide.
  • a transient receptor potential channel polypeptide For example, in one such system Autographa californica nuclear polyhedrosis virus (ACNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding transient receptor potential channel polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of transient receptor potential channel polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which transient receptor potential channel polypeptides can be expressed (Engelhard et al., Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).
  • a number of viral-based expression systems can be used to express transient receptor potential channel polypeptides in mammalian host cells.
  • sequences encoding transient receptor potential channel polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing a transient receptor potential channel polypeptide in infected host cells (Logan & Shenk, Proc. Nat. Acad. Sci. 81, 3655-3659, 1984).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • HACs Human artificial chromosomes
  • 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding transient receptor potential channel polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a transient receptor potential channel polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al., Results Probl. Cell Differ. 20, 125-162, 1994).
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed transient receptor potential channel polypeptide in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a “prepro”form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities e.g., CHO, HeLa, MDCK, HEK293, and WI38
  • ATCC American Type Culture Collection
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express transient receptor potential channel polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced transient receptor potential channel sequences.
  • Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.
  • herpes simplex virus thymidine kinase (Wigler et al., Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al., Cell 22 , 817 -23, 1980) genes which can be employed in tk or aprf cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci.
  • npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988).
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al., Methods Mol. Biol. 55, 121-131, 1995).
  • transient receptor potential channel polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a transient receptor potential channel polypeptide is inserted within a marker gene sequence, transformed cells containing sequences that encode a transient receptor potential channel polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a transient receptor potential channel polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the transient receptor potential channel polynucleotide.
  • host cells which contain a transient receptor potential channel polynucleotide and which express a transient receptor potential channel polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding a transient receptor potential channel polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleo-tides encoding a transient receptor potential channel polypeptide.
  • Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a transient receptor potential channel polypeptide to detect transformants that contain a transient receptor potential channel polynucleotide.
  • a variety of protocols for detecting and measuring the expression of a transient receptor potential channel polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immuno-assay using monoclonal antibodies reactive to two non-interfering epitopes on a transient receptor potential channel polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al., SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al., J. Exp. Med. 158, 12
  • a wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding transient receptor potential channel polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding a transient receptor potential channel polypeptide can be cloned into a vector for the production of an mRNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a transient receptor potential channel polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode transient receptor potential channel polypeptides can be designed to contain signal sequences which direct secretion of soluble transient receptor potential channel polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound transient receptor potential channel polypeptide.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.).
  • cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and the transient receptor potential channel polypeptide also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing a transient receptor potential channel polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al., Prot. Exp.
  • enterokinase cleavage site provides a means for purifying the transient receptor potential channel polypeptide from the fusion protein.
  • Vectors that contain fusion proteins are disclosed in Kroll et al., DNA Cell Bio. 12, 441-453, 1993.
  • transient receptor potential channel polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al., Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980).
  • a transient receptor potential channel polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al., Science 269, 202-204, 1995).
  • Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of transient receptor potential channel polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983).
  • the composition of a synthetic transient receptor potential channel polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the transient receptor potential channel polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fuision protein.
  • transient receptor potential channel polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life that is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter transient receptor potential channel polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • antibody any type of antibody known in the art can be generated to bind specifically to an epitope of a transient receptor potential channel polypeptide.
  • “Antibody” as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab′) 2 , and Fv, which are capable of binding an epitope of a transient receptor potential channel polypeptide.
  • Fab fragment antigen binding domain
  • F(ab′) 2 fragment antigen binding
  • Fv fragments thereof
  • epitope of a transient receptor potential channel polypeptide typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope.
  • epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
  • An antibody which specifically binds to an epitope of a transient receptor potential channel polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.
  • an antibody which specifically binds to a transient receptor potential channel polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to transient receptor potential channel polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a transient receptor potential channel polypeptide from solution.
  • Human transient receptor potential channel polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a transient receptor potential channel polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response.
  • a carrier protein such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).
  • mineral gels e.g., aluminum hydroxide
  • surface active substances e.g lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG bacilli Calmette-Guerin
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies that specifically bind to a transient receptor potential channel polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoina technique (Kohler et al., Nature 256, 495-497, 1985; Kozbor et al., J. Immunol. Methods 81, 31-42, 1985; Cote et al., Proc. Natl. Acad. Sci. 80,2026-2030, 1983; Cole et al., Mol. Cell Biol. 62, 109-120, 1984).
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used Morrison et al., Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al., Nature 312, 604-608, 1984; Takeda et al., Nature 314, 452-454, 1985).
  • Monoclonal and other antibodies also can be “humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues.
  • rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • humanized antibodies can be produced using recombinant methods, as described in GB2188638B.
  • Antibodies that specifically bind to a transient receptor potential channel polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. Pat. No. 5,565,332.
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al., 1996, Eur. J Cancer Prev. 5, 507-11).
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.
  • a nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al., 1995, Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J. Immunol. Meth. 165, 81-91).
  • Antibodies which specifically bind to transient receptor potential channel poly-peptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al., Nature 349, 293-299, 1991).
  • chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the “diabodies” described in WO 94/13804, also can be prepared.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a transient receptor potential channel polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.
  • Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation.
  • an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used.
  • Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of transient receptor potential channel gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5′ end of one nucleotide with the 3′ end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al., Chem. Rev. 90, 543-583,1990.
  • Modifications of transient receptor potential channel gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5′, or regulatory regions of the transient receptor potential channel gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions ⁇ 10and +10 from the start site, are preferred. Similarly, inhibition can be achieved using “triple helix” base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a transient receptor potential channel polynucleotide, each separated by a stretch of contiguous nucleotides which are not. complementary to adjacent transient receptor potential channel nucleotides, can provide sufficient targeting specificity for transient receptor potential channel mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular transient receptor potential channel polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a transient receptor potential channel polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, intemucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3′, 5′-substituted oligonucleotide in which the 3′ hydroxyl group or the 5′ phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al., Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al., Chem. Rev. 90, 543-584, 1990; Uhlmann et al., Tetrahedron Lett. 215, 3539-3542, 1987.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Pat. No. 5,641,673).
  • ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Examples include engineered hanmnerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a transient receptor potential channel polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the transient receptor potential channel polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete “hybridization” region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al., EP 321,201).
  • Specific ribozyme cleavage sites within a transient receptor potential channel RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate transient receptor potential channel RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease transient receptor potential channel expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • genes whose products interact with human transient receptor potential channel may represent genes that are differentially expressed in disorders including, but not limited to, cancer, cardiovascular disorders, and CNS disorders. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human transient receptor potential channel gene or gene product may itself be tested for differential expression.
  • the degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques.
  • standard characterization techniques such as differential display techniques.
  • Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.
  • RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al., ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Pat. No. 4,843,155.
  • Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al., Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al., Nature 308, 149-53; Lee et al., Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and, preferably, differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S. Pat. No. 5,262,311).
  • the differential expression information may itself suggest relevant methods for the treatment of disorders involving the human transient receptor potential channel.
  • treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human transient receptor potential channel.
  • the differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human transient receptor potential channel gene or gene product are up-regulated or down-regulated.
  • the invention provides assays for screening test compounds that bind to or modulate the activity of a transient receptor potential channel polypeptide or a transient receptor potential channel polynucleotide.
  • a test compound preferably binds to a transient receptor potential channel polypeptide or polynucleotide. More preferably, a test compound decreases or increases functional activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinately or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the “one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.
  • Test compounds can be screened for the ability to bind to transient receptor potential channel polypeptides or polynucleotides or to affect transient receptor potential channel activity or transient receptor potential channel gene expression using high throughput screening.
  • high throughput screening many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened.
  • the most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 ⁇ l.
  • many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al., Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994).
  • the cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose.
  • the combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Chelsky “Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches,” reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7- 10, 1995) .
  • Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel.
  • beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • test samples are placed in a porous matrix.
  • One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support.
  • the test compound is preferably a small molecule that binds to the transient receptor potential channel polypeptide such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • either the test compound or the transient receptor potential channel polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound that is bound to the transient receptor potential channel polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • binding of a test compound to a transient receptor potential channel polypeptide can be determined without labeling either of the interactants.
  • a microphysiometer can be used to detect binding of a test compound with a transient receptor potential channel polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • a microphysiometer is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a transient receptor potential channel polypeptide (McConnell et al., Science 257, 1906-1912, 1992).
  • BIA Bimolecular Interaction Analysis
  • a transient receptor potential channel polypeptide can be used as a “bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., Cell 72, 223-232, 1993; Madura et al., J Biol. Chem.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding a transient receptor potential channel polypeptide can be fused to a poly-nucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey” or “sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transciptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the transient receptor potential channel polypeptide.
  • a reporter gene e.g., LacZ
  • transient receptor potential channel poly-nucleotide or polynucleotide
  • test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • any method known in the art can be used to attach the polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a transient receptor potential channel polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • the transient receptor potential channel polypeptide is a fusion protein comprising a domain that allows the transient receptor potential channel polypeptide to be bound to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed transient receptor potential channel polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • transient receptor potential channel polypeptide or polynucleotide
  • test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated transient receptor potential channel polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies which specifically bind to a transient receptor potential channel poly-peptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to the transient receptor potential channel polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the transient receptor potential channel polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Screening for test compounds which bind to a transient receptor potential channel polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a transient receptor potential channel polypeptide or polynucleotide can be used in a cell-based assay system. A transient receptor potential channel poly-nucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a transient receptor potential channel polypeptide or polynucleotide is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease a biological effect of a human transient receptor potential channel. Such biological effects can be determined for example using functional assays such as those described below. Functional assays can be carried out after contacting either a purified transient receptor potential channel polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound which increases or decreases a functional activity of a transient receptor potential channel polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent.
  • Ion channels can be tested functionally in living cells.
  • Polypeptides comprising amino acid sequences encoded by open reading frames of the invention are either expressed endogeneously in appropriate reporter cells or are introduced recombinantly.
  • Channel activity can be monitored by concentration changes of the permeating ion, by changes in the transmembrane electrical potential gradient, or by measuring a cellular response (e.g., expression of a reporter gene or secretion of a neuro-transmitter triggered or modulated by the polypeptide's activity.
  • the activity of ion channel proteins in cells can be determined, for example, by loading the cells with an ion-sensitive fluorescent indicator.
  • Fluorescent indicators can be loaded into cells in 96-well plates or another container, and the activity of ion channel proteins in the presence or absence of various test compounds can be simply and rapidly determined. See, e.g., U.S. Pat. No. 6,057,114.
  • Ion channel currents result in changes of electrical membrane potential (V m ) which can be monitored directly using potentiometric fluorescent probes.
  • These electrically charged indicators e.g., the anionic oxonol dye DiBAC 4 (3)
  • transient receptor potential channel Another approach to determining the activity of ion channel proteins involves the electrophysiological determination of ionic currents.
  • Cells which endogenously express a transient receptor potential channel can be used to study the effects of various test compounds or transient receptor potential channel polypeptides on endogenous ionic currents attributable to the activity of transient receptor potential channels.
  • cells which do not express transient receptor potential channel can be employed as hosts for the expression of transient receptor potential channel, whose activity can then be studied by electrophysiological or other means.
  • Cells preferred as host cells for the heterologous expression of transient receptor potential channel are preferably mammalian cells such as COS cells, mouse L cells, CHO cells (e.g., DG44 cells), human embryonic kidney cells (e.g., HEK293 cells), African green monkey cells and the like; amphibian cells, such as Xenopus laevis oocytes; or cells of yeast such as S. cerevisiae or P. pastoris . See, e.g., U.S. Pat. No. 5,876,958.
  • mammalian cells such as COS cells, mouse L cells, CHO cells (e.g., DG44 cells), human embryonic kidney cells (e.g., HEK293 cells), African green monkey cells and the like
  • amphibian cells such as Xenopus laevis oocytes
  • yeast such as S. cerevisiae or P. pastoris . See, e.g., U.S. Pat. No. 5,876,958
  • Electrophysiological procedures for measuring the current across a cell membrane are well known.
  • a preferred method is the use of a voltage clamp as in the whole-cell patch clamp technique.
  • Non-calcium currents can be eliminated by established methods so as to isolate the ionic current flowing through ion channel proteins.
  • ionic currents resulting from endogenous ion channel proteins can be suppressed by known pharmacological or electrophysiological techniques. See, e.g., U.S. Pat. No. 5,876,958.
  • a further activity of the transient receptor potential channel which can be assessed is its ability to bind various ligands, including test compounds.
  • the ability of a test compound to bind transient receptor potential channel or fragments thereof may be determined by any appropriate competitive binding analysis (e.g., Scatchard plots), wherein the binding capacity and/or affinity is determined in the presence and absence of one or more concentrations a compound having known affinity for the transient receptor potential channel. Binding assays can be performed using whole cells that express transient receptor potential channel (either endogenously or heterologously), membranes prepared from such cells, or purified transient receptor potential channel.
  • test compounds that increase or decrease transient receptor potential channel gene expression are identified.
  • a transient receptor potential channel polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the transient receptor potential channel polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of transient receptor potential channel mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of a transient receptor potential channel polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a transient receptor potential channel polypeptide.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell that expresses a transient receptor potential channel polynucleotide can be used in a cell-based assay system.
  • the transient receptor potential channel polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above.
  • Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.
  • compositions of the invention can comprise, for example, a transient receptor potential channel polypeptide, transient receptor potential channel polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a transient receptor potential channel polypeptide, or mimetics, activators, or inhibitors of a transient receptor potential channel polypeptide activity.
  • the compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.
  • Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers also can be used for delivery.
  • the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1% -2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • TRPC Human transient receptor potential channel
  • MTR1 is expressed in many rhabdomyosarcomas and Wilm's tumors, suggesting a role of TRPCs in tumor growth (6). Mutations in TRPCs are found in patients who have mucolipidosis type IV, which is a neurodegenerative disorder. Thus, it is believed that the human transient receptor potential channel of the invention can be regulated to treat cancer, cardiovascular disorders, and CNS disorders.
  • Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Various combinations of these aberrant physiologies, along with the acquisition of drug-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue.
  • Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins. These proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities. Activators and/or inhibitors of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drug development and subsequent testing in humans.
  • Cardiovascular diseases include the following disorders of the heart and the vascular system: congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, and peripheral vascular diseases.
  • Heart failure is defined as a pathophysiologic state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failure, such as high-output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.
  • MI Myocardial infarction
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen.
  • This group of diseases includes stable angina, unstable angina, and asymptomatic ischemia.
  • Arrhythmias include all forms of atrial and ventricular tachyarrhythmias (atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexcitation syndrome, ventricular tachycardia, ventricular flutter, and ventricular fibrillation), as well as bradycardic forms of arrhythmias.
  • Vascular diseases include primary as well as all kinds of secondary arterial hypertension (renal, endocrine, neurogenic, others).
  • the disclosed gene and its product may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications.
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon, and venous disorders.
  • PAOD peripheral arterial occlusive disease
  • acute arterial thrombosis and embolism inflammatory vascular disorders
  • Raynaud's phenomenon Raynaud's phenomenon
  • venous disorders venous disorders.
  • Central and peripheral nervous system disorders also can be treated, such as primary and secondary disorders after brain injury, disorders of mood, anxiety disorders, disorders of thought and volition, disorders of sleep and wakefulness, diseases of the motor unit, such as neurogenic and myopathic disorders, neurodegenerative disorders such as Alzheimer's and Parkinson's disease, and processes of peripheral and chronic pain.
  • Pain that is associated with CNS disorders also can be treated by regulating the activity of human transient receptor potential channel. Pain which can be treated includes that associated with central nervous system disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
  • central nervous system disorders such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation).
  • Non-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania.
  • headache pain for example, migraine with aura, migraine without aura, and other migraine disorders
  • episodic and chronic tension-type headache tension-type like headache, cluster headache, and chronic par
  • This invention flier pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a transient receptor potential channel polypeptide binding molecule
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • a reagent which affects transient receptor potential channel activity can be administered to a human cell, either in vitro or in vivo, to reduce transient receptor potential channel activity.
  • the reagent preferably binds to an expression product of a human transient receptor potential channel gene. If the expression product is a protein, the reagent is preferably an antibody.
  • an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.
  • the reagent is delivered using a liposome.
  • the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours.
  • a liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human.
  • the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.
  • a liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma mernbrane of the targeted cell to deliver its contents to the cell.
  • the transfection efficiency of a liposome is about 0.5 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, more preferably about 1.0 ⁇ g of DNA per 16 nmole of liposome delivered to about 10 6 cells, and even more preferably about 2.0 ⁇ g of DNA per 16 nmol of liposome delivered to about 10 6 cells.
  • a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol.
  • a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.
  • a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Pat. No. 5,705,151).
  • a reagent such as an antisense oligonucleotide or ribozyme
  • antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery.
  • Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993); Chiou et al., GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al., J. Biol. Chem. 269, 542-46 (1994); Zenke et al., Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al., J. Biol. Chem. 266, 338-42 (1991).
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases transient receptor potential channel activity relative to the transient receptor potential channel activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
  • the animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 .
  • compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • the reagent is a single-chain antibody
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, “gene gun,” and DEAE- or calcium phosphate-mediated transfection.
  • Effective in vivo dosages of an antibody are in the range of about 5 ⁇ g to about 50 ⁇ g/kg, about 50 ⁇ g to about 5 mg/kg, about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight, and about 200 to about 250 ⁇ g/kg of patient body weight.
  • effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA.
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of a transient receptor potential channel gene or the activity of a transient receptor potential channel polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of a transient receptor potential channel gene or the activity of a transient receptor potential channel polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to transient receptor potential channel-specific mRNA, quantitative RT-PCR, immunologic detection of a transient receptor potential channel polypeptide, or measurement of transient receptor potential channel activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatmnent or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Human transient receptor potential channel also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the polypeptide. For example, differences can be determined between the cDNA or genomic sequence encoding transient receptor potential channel in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method.
  • cloned DNA segments can be employed as probes to detect specific DNA segments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.
  • DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science 230, 1242, 1985).
  • Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. U.S.A 85, 4397-4401, 1985).
  • nuclease protection assays such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. U.S.A 85, 4397-4401, 1985).
  • the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA.
  • mutations can also be detected by in situ analysis.
  • Altered levels of transient receptor potential channel also can be detected in various tissues.
  • Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.
  • the polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-transient receptor potential channel polypeptide obtained is transfected into human embryonic kidney 293 cells. 2 days after transfection, cells attached to coverslip are washed twice with HPSS 8120 ml mM NaCl, 5.3 mM KC1, 0.8 mM CaC12, 11.1 mM glucose, 20 mM Hepes (pH 7.4) and loaded with fura 2-AM (Molecular Probes, 5 ⁇ m in HPSS) for 30 minutes at room temperature in the dark.
  • HPSS 8120 ml mM NaCl, 5.3 mM KC1, 0.8 mM CaC12, 11.1 mM glucose, 20 mM Hepes (pH 7.4)
  • fura 2-AM Molecular Probes, 5 ⁇ m in HPSS
  • the coverslips are inserted into a cicular open-bottom chamber and placed onto the stage of a Zeiss Axovert microscope fitted with an Attofluor Digital Imaging and Photometry System(Attofluor Inc., Rockville, Md.) 20-30 isolated fura 2-loaded cells are selected and [Ca2+] i in these cells is measured by1 fluorescence videomicroscopy at room temperature using alternating excitation wavelengths of 334 and 380 nm and monitoring emitted fliorescence at 520 nm. Free [Ca2+] i is calculated from 334/380 fluorescence ratios following the method described previously. All reagents are diluted to their final concentrations in HPSS and applied to the cells by surface perfusion. It is shown that the polypeptide of SEQ ID NO: 2 has a transient receptor potential channel activity.
  • the Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, Calif.) is used to produce large quantities of recombinant human transient receptor potential channel polypeptides in yeast.
  • the transient receptor potential channel-encoding DNA sequence is derived from SEQ ID NO:1.
  • the DNA sequence is modified by well known methods in such a way that it contains at its 5′-end an initiation codon and at its 3′-end an enterokinase cleavage site, a His6 reporter tag and a termination codon.
  • the yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea.
  • the bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, Calif.) according to manufacturer's instructions. Purified human transient receptor potential channel polypeptide is obtained.
  • Transient receptor potential channel polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution.
  • Human transient receptor potential channel polypeptides comprise the amino acid sequence shown in SEQ ID NO:2.
  • the test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound
  • the buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a transient receptor potential channel polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a transient receptor potential channel polypeptide.
  • test compound is administered to a culture of human cells transfected with a transient receptor potential channel expression construct and incubated at 37° C. for 10 to 45 minutes.
  • a culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.
  • RNA is isolated from the two cultures as described in Chirgwin et al., Biochem. 18, 5294-99, 1979).
  • Northern blots are prepared using 20 to 30 ⁇ g total RNA and hybridized with a 32 P-labeled transient receptor potential channel-specific probe at 65° C. in Express-hyb (CLONTECH).
  • the probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO:1.
  • a test compound that decreases the transient receptor potential channel-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of transient receptor potential channel gene expression.
  • RT-PCR Reverse Transcription-Polymerase Chain Reaction
  • transient receptor potential channel is involved in CNS disorders
  • tissues are screened: fetal and adult brain, muscle, heart, lung, kidney, liver, thymus, testis, colon, placenta, trachea, pancreas, kidney, gastric mucosa, colon, liver, cerebellum, skin, cortex (Alzheimer's and normal), hypothalamus, cortex, amygdala, cerebellum, hippocampus, choroid, plexus, thalamus, and spinal cord.
  • transient receptor potential channel is involved in cancer
  • expression is determined in the following tissues: adrenal gland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, uterus, and peripheral blood lymphocytes.
  • Expression in the following cancer cell lines also is determined: DU-145 (prostate), NCI-H125 (lung), HT-29 (colon), COLO-205 (colon), A-549 (lung), NCI-H460 (lung), HT-116 (colon), DLD-1 (colon), MDA-MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435 (breast), HT-1080, MCF-7 (breast), and U87. Matched pairs of malignant and normal tissue from the same patient also are tested.
  • Quantitative expression profiling is performed by the form of quantitative PCR analysis called “kinetic analysis” firstly described in Higuchi et al., BioTechnology 10, 413-17, 1992, and Higuchi et al., BioTechnology 11, 1026-30, 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies.
  • the probe is cleaved by the 5′-3′ endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al., Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al., Genome Res. 6, 986-94, 1996, and Gibson et al., Genome Res. 6, 995-1001, 1996).
  • the amplification of an endogenous control can be performed to standardize the amount of sample RNA added to a reaction.
  • the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous control can be independently quantified in the same tube if probes labeled with different dyes are used.
  • RNA extraction and cDNA preparation Total RNA from the tissues listed above are used for expression quantification. RNAs labeled “from autopsy” were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, Md.) according to the manufacturer's protocol.
  • RNA Fifty ⁇ g of each RNA were treated with DNase I for 1 hour at 37 C in the following reaction mix: 0.2 U/ ⁇ l RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/ ⁇ l RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; 10mM MgCl 2; 50 mM NaCl; and 1 mM DTT.
  • RNA is extracted once with 1 volume of phenol:chloroform:isoamyl alcohol (24:24:1) and once with chloroform, and precipitated with ⁇ fraction (1/10) ⁇ volume of 3 M NaAcetate, pH5.2, and 2 volumes of ethanol.
  • RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, Tex.). After resuspension and spectrophotometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200 ng/ ⁇ L. Reverse transcription is carried out with 2.5 ⁇ M of random hexamer primers.
  • TaqMan quantitative analysis Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5′ end FAM (6-carboxy-fluorescein) and at the 3′ end with TAMRA (6-carboxy-tetramethyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.
  • FAM 6-carboxy-fluorescein
  • TAMRA 6-carboxy-tetramethyl-rhodamine
  • Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).
  • PDAR Pre-Developed TaqMan Assay Reagents
  • the assay reaction mix is as follows: 1 ⁇ final TaqMan Universal PCR Master Mix (from 2 ⁇ stock) (PE Applied Biosystems, CA); 1 ⁇ PDAR control—18S RNA (from 20 ⁇ stock); 300 nM forward primer; 900 nM reverse primer; 200 nM probe; 10 ng cDNA; and water to 25 ⁇ l.
  • the experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA).
  • fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity.
  • This non-tumor assay measures the ability of a compound to reduce either the endogenous level of a circulating hormone or the level of hormone produced in response to a biologic stimulus.
  • Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.).
  • test compound p.o., i.p., i.v., i.m., or s.c.
  • Plasma is assayed for levels of the hormone of interest. If the normal circulating levels of the hormone are too low and/or variable to provide consistent results, the level of the hormone may be elevated by a pre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m.
  • a biologic stimulus i.e., LHRH may be injected i.m.
  • mice were fed at a dosage of 30 ng/mouse to induce a burst of testosterone synthesis).
  • the timing of plasma collection would be adjusted to coincide with the peak of the induced hormone response.
  • Compound effects are compared to a vehicle-treated control group.
  • An F-test test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol, these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as compared to the vehicle control group.
  • specific readout assay protocol these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as
  • a hormone dependent tissue i.e., seminal vesicles in males and uteri in females.
  • Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.) according to a predetermined schedule and for
  • Organ weights may be directly compared or they may be normalized for the body weight of the animal. Compound effects are compared to a vehicle-treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol. Cell proliferation is determined by measuring a marker of cell number (i.e., MTT or LDH). The cell number and change in cell number from the starting inoculum are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p ⁇ 0.05 as compared to the vehicle control group.
  • a marker of cell number i.e., MTT or LDH
  • Hydron pellets with or without growth factors or cells are implanted into a micropocket surgically created in the rodent cornea.
  • Compound administration may be systemic or local (compound mixed with growth factors in the hydron pellet).
  • Corneas are harvested at 7 days post implantation immediately following intracardiac infusion of colloidal carbon and are fixed in 10% formalin. Readout is qualitative scoring and/or image analysis. Qualitative scores are compared by Rank Sum test. Image analysis data is evaluated by measuring the area of neovascularization (in pixels) and group averages are compared by Student's t-test (2 tail). Significance is p ⁇ 0.05 as compared to the growth factor or cells only group.
  • Matrigel containing cells or growth factors, is injected subcutaneously. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Matrigel plugs are harvested at predetermined time point(s) and prepared for readout. Readout is an ELISA-based assay for hemoglobin concentration and/or histological examination (i.e. vessel count, special staining for endothelial surface markers: CD31, factor-8). Readouts are analyzed by Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p ⁇ 0.05 as compared to the vehicle control group.
  • Tumor cells or frgments are implanted subcutaneously on Day 0.
  • Vehicle and/or compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting at a time, usually on Day 1, prior to the ability to measure the tumor burden.
  • Body weights and tumor measurements are recorded 2-3 times weekly. Mean net body and tumor weights are calculated for each data collection day.
  • Anti-tumor efficacy may be initially determined by comparing the size of treated (T) and control (C) tumors on a given day by a Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p ⁇ 0.05.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meirer curves from the times for individual tumors to attain the evaluation size. Significance is p ⁇ 0.05.
  • Tumor cells are injected intraperitoneally or intracranially on Day 0.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting on Day 1. Observations of morbidity and/or mortality are recorded twice daily. Body weights are measured and recorded twice weekly. Morbidity/mortality data is expressed in terms of the median time of survival and the number of long-term survivors is indicated separately. Survival times are used to generate Kaplan-Meier curves. Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment.
  • Tumor cells or fragments are implanted subcutaneously and grown to the desired size for treatment to begin. Once at the predetermined size range, mice are randomized into treatment groups. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • Tumor cells or fragments, of mammary adenocarcinoma origin are implanted directly into a surgically exposed and reflected mammary fat pad in rodents. The fat pad is placed back in its original position and the surgical site is closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay.
  • Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size.
  • Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value ⁇ 0.05 compared to the vehicle control group.
  • this model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ, or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells or fragments, of prostatic adenocarcinoma origin are implanted directly into a surgically exposed dorsal lobe of the prostate in rodents.
  • the prostate is externalized through an abdominal incision so that the tumor can be implanted specifically in the dorsal lobe while verifying that the implant does not enter the seminal vesicles.
  • the successfully inoculated prostate is replaced in the abdomen and the incisions through the abdomen and skin are closed.
  • Hormones may also be administered to the rodents to support the growth of the tumors.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule.
  • Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected.
  • the size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
  • An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor.
  • Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the lungs), or measuring the target organ weight (i.e., the regional lymph nodes). The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells of pulmonary origin may be implanted intrabronchially by making an incision through the skin and exposing the trachea The trachea is pierced with the beveled end of a 25 gauge needle and the tumor cells are inoculated into the main bronchus using a flat-ended 27 gauge needle with a 90° bend.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected.
  • the size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
  • An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group.
  • This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the contralateral lung), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells of gastrointestinal origin may be implanted intracecally by making an abdominal incision through the skin and externalizig the intestine. Tumor cells are inoculated into the cecal wall without penetrating the lumen of the intestine using a 27 or 30 gauge needle. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope.
  • An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p ⁇ 0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the liver), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p ⁇ 0.05 compared to the control group in the experiment.
  • Tumor cells are inoculated s.c. and the tumors allowed to grow to a predetermined range for spontaneous metastasis studies to the lung or liver. These primary tumors are then excised. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule which may include the period leading up to the excision of the primary tumor to evaluate therapies directed at inhibiting the early stages of tumor metastasis. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined.
  • Tumor cells are injected into the tail vein, portal vein, or the left ventricle of the heart in experimental (forced) lung, liver, and bone metastasis studies, respectively.
  • Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves.
  • Significance is p ⁇ 0.05 by a log-rank test compared to the control group in the experiment.
  • the mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance at p ⁇ 0.05 compared to the vehicle control group in the experiment for both endpoints.
  • the cell line used for testing is the human colon cancer cell line HCT116.
  • Cells are cultured in RPMI-1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml and kept at 37° C. in a 95% air/5%CO 2 atmosphere.
  • Phosphorothioate oligoribonucleotides are synthesized on an Applied Biosystems Model 380B DNA synthesizer using phosphoroamnidite chemistry. A sequence of 24 bases complementary to the nucleotides at position 1 to 24 of SEQ ID NO:1 is used as the test oligonucleotide. As a control, another (random) sequence is used: 5′-TCA ACT GAC TAG ATG TAC ATG GAC-3′. Following assembly and deprotection, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate buffered saline at the desired concentration.
  • oligonucleotides Purity of the oligonucleotides is tested by capillary gel electrophoresis and ion exchange HPLC. The purified oligonucleotides are added to the culture medium at a concentration of 10 ⁇ M once per day for seven days.
  • test oligonucleotide for seven days results in significantly reduced expression of human transient receptor potential channel as determined by Western blotting. This effect is not observed with the control oligonucleotide.
  • the number of cells in the cultures is counted using an automatic cell counter. The number of cells in cultures treated with the test oligonucleotide (expressed as 100%) is compared with the number of cells in cultures treated with the control oligonucleotide. The number of cells in cultures treated with the test oligonucleotide is not more than 30% of control, indicating that the inhibition of human transient receptor potential channel has an anti-proliferative effect on cancer cells.
  • Acute pain is measured on a hot plate mainly in rats.
  • Two variants of hot plate testing are used: In the classical variant animals are put on a hot surface (52 to 56° C.) and the latency time is measured until the animals show nocifensive behavior, such as stepping or foot licking.
  • the other variant is an increasing temperature hot plate where the experimental animals are put on a surface of neutral temperature. Subsequently this surface is slowly but constantly heated until the animals begin to lick a hind paw. The temperature which is reached when hind paw licking begins is a measure for pain threshold.
  • Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal
  • Persistent pain is measured with the formalin or capsaicin test, mainly in rats.
  • a solution of 1 to 5% formalin or 10 to 100 ⁇ g capsaicin is injected into one hind paw of the experimental animal.
  • the animals show nocifensive reactions like flinching, licking and biting of the affected paw.
  • the number of nocifensive reactions within a time frame of up to 90 minutes is a measure for intensity of pain.
  • Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradernal, transdermal) prior to formalin or capsaicin administration.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c., intradernal, transdermal
  • Neuropathic pain is induced by different variants of unilateral sciatic nerve injury mainly in rats.
  • the operation is performed under anesthesia.
  • the first variant of sciatic nerve injury is produced by placing loosely constrictive ligatures around the common sciatic nerve.
  • the second variant is the tight ligation of about the half of the diameter of the common sciatic nerve.
  • a group of models is used in which tight ligations or transections are made of either the L5 and L6 spinal nerves, or the L% spinal nerve only.
  • the fourth variant involves an axotomy of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact whereas the last variant comprises the axotomy of only the tibial branch leaving the sural and common nerves uninjured. Control animals are treated with a sham operation.
  • the nerve injured animals develop a chronic mechanical allodynia, cold allodynioa, as well as a thermal hyperalgesia
  • Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc.-Life Science Instruments, Woodland Hills, SA, USA; Electronic von Frey System, Somedic Sales AB, Hörby, Sweden).
  • Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy), or by means of a cold plate of 5 to 10° C. where the nocifensive reactions of the affected hind paw are counted as a measure of pain intensity.
  • a further test for cold induced pain is the counting of nocifensive reactions, or duration of nocifensive responses after plantar administration of acetone to the affected hind limb.
  • Chronic pain in general is assessed by registering the circadanian rhythms in activity (Surjo and Arndt, (2015) zu GmbH, Cologne, Germany), and by scoring differences in gait (foot print patterns; FOOTPRINTS program, Klapdor et al., 1997. A low cost method to analyze footprint patterns. J. Neurosci. Methods 75, 49-54).
  • Compounds are tested against sham operated and vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal
  • Inflammatory pain is induced mainly in rats by injection of 0.75 mg carrageenan or complete Freund's adjuvant into one hind paw.
  • the animals develop an edema with mechanical allodynia as well as thermal hyperalgesia.
  • Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc.-Life Science Instruments, Woodland Hills, SA, USA).
  • Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy, Paw thermal stimulator, G. Ozaki, University of California, USA).
  • Plant Test Ugo Basile, Comerio, Italy
  • Paw thermal stimulator G. Ozaki, University of California, USA
  • Compounds are tested against uninflamed as well as vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal
  • Compounds are tested against diabetic and non-diabetic vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing.
  • application routes i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal
  • Degeneration of the dopaminergic nigrostriatal and striatopallidal pathways is the central pathological event in Parkinson's disease. This disorder has been mimicked experimnentally in rats using single/sequential unilateral stereotaxic injections of 6-OH-DA into the medium forebrain bundle (MFB).
  • MFB medium forebrain bundle
  • Animals are administered pargyline on the day of surgery (Sigma, St. Louis, Mo., USA; 50 mg/kg i.p.) in order to inhibit metabolism of 6-OHDA by monoamine oxidase and desmethyliiipramine HCl (Sigma; 25 mg/kg i.p.) in order to prevent uptake of 6-OHDA by noradrenergic terminals. Thirty minutes later the rats are anesthetized with sodium pentobarbital (50 mg/kg) and placed in a stereotaxic frame.
  • DA nigrostriatal pathway 4 ⁇ l of 0.01% ascorbic acid-saline containing 8 ⁇ g of 6-OHDA HBr (Sigma) are injected into the left medial fore-brain bundle at a rate of 1 ⁇ l/min (2.4 mm anterior, 1.49 mm lateral, ⁇ 2.7 mm ventral to Bregma and the skull surface). The needle is left in place an additional 5 min to allow diffusion to occur.
  • Forelimb akinesia is assessed three weeks following lesion placement using a modified stepping test protocol.
  • the animals are held by the experimenter with one hand fixing the hindlimbs and slightly raising the hind part above the surface.
  • One paw is touching the table, and is then moved slowly sideways (5 s for 1 m), first in the forehand and then in the backhand direction.
  • the number of adjusting steps is counted for both paws in the backhand and forehand direction of movement.
  • the sequence of testing is right paw forehand and backhand adjusting stepping, followed by left paw forehand and backhand directions.
  • the test is repeated three times on three consecutive days, after an initial training period of three days prior to the first testing.
  • Forehand adjusted stepping reveals no consistent differences between lesioned and healthy control animals. Analysis is therefore restricted to backhand adjusted stepping.
  • a modified version of the staircase test is used for evaluation of paw reaching behavior three weeks following primary and secondary lesion placement.
  • Plexiglass test boxes with a central platform and a removable staircase on each side are used.
  • the apparatus is designed such that only the paw on the same side at each staircase can be used, thus providing a measure of independent forelimb use.
  • For each test the animals are left in the test boxes for 15 min.
  • the double staircase is filled with 7 ⁇ 3 chow pellets (Precision food pellets, formula: P, purified rodent diet, size 45 mg; Sandown Scientific) on each side.
  • MPTP neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine
  • DAergic mesencephalic dopaminergic
  • TH tyrosine hydroxylase
  • mice are perfused transcardially with 0.01 M PBS (pH 7.4) for 2 min, followed by 4% paraformaldehyde (Merck) in PBS for 15 min.
  • the brains are removed and placed in 4% paraformaldehyde for 24 h at 4° C. For dehydration they are then transferred to a 20% sucrose (Merck) solution in 0.1 M PBS at 4° C. until they sink.
  • the brains are frozen in methylbutan at ⁇ 20° C. for 2 min and stored at ⁇ 70° C.
  • sledge microtome (mod. 3800-Frigocut, Leica) 25 ⁇ m sections are taken from the genu of the corpus callosum (AP 1.7 mm) to the hippocampus (AP 21.8 mm) and from AP 24.16 to AP 26.72. Forty-six sections are cut and stored in assorters in 0.25 M Tris buffer (pH 7.4) for immunohistochernistry.
  • TH free-floating tyrosine hydroxylase
  • the system logs the fall as the end of the experiment for that mouse, and the total time on the rotarod, as well as the time of the fall and all the set-up parameters, are recorded.
  • the system also allows a weak current to be passed through the base grid, to aid training.
  • the object recognition task has been designed to assess the effects of experimental manipulations on the cognitive performance of rodents.
  • a rat is placed in an open field, in which two identical objects are present.
  • the rats inspects both objects during the first trial of the object recognition task.
  • a second trial after a retention interval of for example 24 hours, one of the two objects used in the first trial, the ‘familiar’ object, and a novel object are placed in the open field.
  • the inspection time at each of the objects is registered.
  • the basic measures in the OR task is the time spent by a rat exploring the two object the second trial. Good retention is reflected by higher exploration times towards the novel than the ‘familiar’ object.
  • Administration of the putative cognition enhancer prior to the first trial predominantly allows assessment of the effects on acquisition, and eventually on consolidation processes.
  • Administration of the testing compound after the first trial allows to assess the effects on consolidation processes, whereas administration before the second trial allows to measure effects on retrieval processes.
  • the passive avoidance task assesses memory performance in rats and mice.
  • the inhibitory avoidance apparatus consists of a two-compartment box with a light compartment and a dark compartment. The two compartments are separated by a guillotine door that can be operated by the experimenter. A threshold of 2 cm separates the two compartments when the guillotine door is raised. When the door is open, the illumination in the dark compartment is about 2 lux. The light intensity is about 500 lux at the center of the floor of the light compartment.
  • the step-through latency that is the first latency of entering the dark compartment (in sec.) during the retention session is an index of the memory performance of the animal; the longer the latency to enter the dark compartment, the better the retention is.
  • the Morris water escape task measures spatial orientation learning in rodents. It is a test system that has extensively been used to investigate the effects of putative therapeutic on the cognitive functions of rats and mice. The performance of an animal is assessed in a circular water tank with an escape platform that is submerged about 1 cm below the surface of the water. The escape platform is not visible for an animal swimming in the water tank. Abundant extra-maze cues are provided by the furniture in the room, including desks, computer equipment, a second water tank, the presence of the experimenter, and by a radio on a shelf that is playing softly.
  • the animals receive four trials during five daily acquisition sessions.
  • a trial is started by placing an animal into the pool, facing the wall of the tank.
  • Each of four starting positions in the quadrants north, east, south, and west is used once in a series of four trials; their order is randomized.
  • the escape platform is always in the same position.
  • a trial is terminated as soon as the animal had climbs onto the escape platform or when 90 seconds have elapsed, whichever event occurs first.
  • the animal is allowed to stay on the platform for 30 seconds. Then it is taken from the platform and the next trial is started. If an animal did not find the platform within 90 seconds it is put on the platform by the experimenter and is allowed to stay there for 30 seconds.
  • an additional trial is given as a probe trial: the platform is removed, and the time the animal spends in the four quadrants is measured for 30 or 60 seconds.
  • the probe trial all animals start from the same start position, opposite to the quadrant where the escape platform had been positioned during acquisition.
  • the T-maze spontaneous alternation task assesses the spatial memory performance in mice.
  • the start arm and the two goal arms of the T-maze are provided with guillotine doors which can be operated manually by the experimenter.
  • a mouse is put into the start arm at the beginning of training.
  • the guillotine door is closed.
  • the ‘forced trial’ either the left or right goal arm is blocked by lowering the guillotine door.
  • the mouse After the mouse has been released from the start arm, it will negotiate the maze, eventually enter the open goal arm, and return to the start position, where it will be confined for 5 seconds, by lowering the guillotine door.
  • the animal can choose freely between the left and right goal arm (all guillotine-doors opened) during 14 ‘free choice’ trials. As soon a the mouse has entered one goal arm, the other one is closed. The mouse eventually returns to the start arm and is free to visit whichever go alarm it wants after having been confined to the start arm for 5 seconds. After completion of 14 free choice trials in one session, the animal is removed from the maze. During training, the animal is never handled.
  • the percent alternations out of 14 trials is calculated. This percentage and the total time needed to complete the first forced trial and the subsequent 14 free choice trials (in s) is analyzed. Cognitive deficits are usually induced by an injection of scopolamine, 30 min before the start of the training session. Scopolamine reduced the per-cent alternations to chance level, or below. A cognition enhancer, which is always administered before the training session, will at least partially, antagonize the scopolamine-induced reduction in the spontaneous alternation rate.
  • TRP4 human transient receptor potential 4
  • TRP4 human transient receptor potential 4
  • Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel.
  • MIR1 melastatin 1
  • trp gene family located in the BWS-WT2 critical region on chromosome 11p15.5 and showing allele-specific expression.
  • Prawitt D Enklaar T
  • Klemm G Gartner B
  • Spangenberg C Winterpacht A
  • Higgins M Higgins M
  • Pelletier J Zabel B

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Abstract

Reagents which regulate human transient receptor potential channel and reagents which bind to human transient receptor potential channel gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, cancer, cardiovascular disorders, and CNS disorders.

Description

    TECHNICAL FIELD OF THE INVENTION
  • The invention relates to the area of ion channel regulation. [0001]
    • BACKGROUND OF THE INVENTION
  • Ion channels are integral membrane proteins, typically comprising multiple subunits, which form selective and highly regulated pores in cellular membranes. Each of these pores controls the influx and efflux of a given ion (e.g., sodium, potassium, calcium, or chloride) across the plasma membrane or the membranes of intracellular compartmnents. Many important physiological processes depend on the control of ion gradients by ion channels. Such processes include synaptic transmission, secretion, fertilization, muscle contraction, and regulation of intracellular and extracellular ion concentrations and pH. Ion channels open in response to various stimuli. For example, there are ligand-gated channels, second messenger-gated channels, voltage-gated channels, and shear- or stress-gated channels. Certain channels allow ions to leak across membranes without a specific stimulus. The gating properties characteristic of a given channel include the period of time it is open, the frequency of opening, the strength of stimulus required for activation, and the refractory period. These characteristics can vary depending on the subunit composition of the channel, association of the channel with accessory proteins, and phosphorylation or other post-translational modification of channel polypeptides. See, e.g., U.S. Pat. No. 6,071,720. [0002]
  • Because of the important biological effects of ion channel proteins, there is a need in the art to identify additional channels whose activity can be regulated to provide therapeutic effects. [0003]
    • SUMMARY OF THE INVENTION
  • It is an object of the invention to provide reagents and methods of regulating a human transient receptor potential channel. This and other objects of the invention are provided by one or more of the embodiments described below. [0004]
  • One embodiment of the invention is a transient receptor potential channel polypeptide comprising an amino acid sequence selected from the group consisting of: [0005]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 2; [0006]
  • the amino acid sequence shown in SEQ ID NO: 2; [0007]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 11; [0008]
  • the amino acid sequence shown in SEQ ID NO:11; [0009]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 12; and [0010]
  • the amino acid sequence shown in SEQ ID NO: 12. [0011]
  • Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a transient receptor potential channel polypeptide comprising an amino acid sequence selected from the group consisting of: [0012]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 2; [0013]
  • the amino acid sequence shown in SEQ ID NO: 2; [0014]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 11; [0015]
  • the amino acid sequence shown in SEQ ID NO:11; [0016]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 12; and [0017]
  • the amino acid sequence shown in SEQ ID NO:12. [0018]
  • Binding between the test compound and the transient receptor potential channel poly-peptide is detected. A test compound which binds to the transient receptor potential channel polypeptide is thereby identified as a potential agent for decreasing extra-cellular matrix degradation. The agent can work by decreasing the activity of the transient receptor potential channel. [0019]
  • Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a polynucleotide encoding a transient receptor potential channel polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: [0020]
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; [0021]
  • the nucleotide sequence shown in SEQ ID NO: 1; [0022]
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 9; [0023]
  • the nucleotide sequence shown in SEQ ID NO: 9; [0024]
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10; and [0025]
  • the nucleotide sequence shown in SEQ ID NO:10. [0026]
  • Binding of the test compound to the polynucleotide is detected. A test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the amount of the transient receptor potential channel through interacting with the transient receptor potential channel mRNA. [0027]
  • Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation. A test compound is contacted with a transient receptor potential channel polypeptide comprising an amino acid sequence selected from the group consisting of: [0028]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 2; [0029]
  • the amino acid sequence shown in SEQ ID NO: 2; [0030]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 11; [0031]
  • the amino acid sequence shown in SEQ ID NO:11; [0032]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 12; and [0033]
  • the amino acid sequence shown in SEQ ID NO: 12. [0034]
  • A transient receptor potential channel activity of the polypeptide is detected. A test compound which increases transient receptor potential channel activity of the polypeptide relative to transient receptor potential channel activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix degradation. A test compound which decreases transient receptor potential channel activity of the polypeptide relative to transient receptor potential channel activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation. [0035]
  • Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a transient receptor potential channel product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of: [0036]
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; [0037]
  • the nucleotide sequence shown in SEQ ID NO: 1; [0038]
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 9; [0039]
  • the nucleotide sequence shown in SEQ ID NO: 9; [0040]
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10; and [0041]
  • the nucleotide sequence shown in SEQ ID NO:10. [0042]
  • Binding of the test compound to the transient receptor potential channel product is detected. A test compound which binds to the transient receptor potential channel product is thereby identified as a potential agent for decreasing extracellular matrix degradation. [0043]
  • Still another embodiment of the invention is a method of reducing extracellular matrix degradation. A cell is contacted with a reagent which specifically binds to a polynucleotide encoding a transient receptor potential channel polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of: [0044]
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; [0045]
  • the nucleotide sequence shown in SEQ ID NO: 1; [0046]
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 9; [0047]
  • the nucleotide sequence shown in SEQ ID NO: 9; [0048]
  • nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 10; and [0049]
  • the nucleotide sequence shown in SEQ ID NO:10. [0050]
  • Transient receptor potential channel activity in the cell is thereby decreased. [0051]
  • The invention thus provides a human transient receptor potential channel that can be used to identify test compounds that may act, for example, as activators or inhibitors of human transient receptor potential channel. Human transient receptor potential channel and fragments thereof also are useful in raising specific antibodies that can block the polypeptide and effectively reduce its activity.[0052]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:1). [0053]
  • FIG. 2 shows the amino acid sequence deduced from the DNA-sequence of FIG. 1 (SEQ ID NO:2). [0054]
  • FIG. 3 shows the amino acid sequence of a protein identified by swissnew|O94759|TRP7_HUMAN TRANSIENT RECEPTOR POTENTIAL CHANNEL7 (SEQ ID NO:3). [0055]
  • FIG. 4 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:4). [0056]
  • FIG. 5 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:5). [0057]
  • FIG. 6 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:6). [0058]
  • FIG. 7 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:7). [0059]
  • FIG. 8 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO 8:). [0060]
  • FIG. 9 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:9). [0061]
  • FIG. 10 shows the DNA-sequence encoding a transient receptor potential channel Polypeptide (SEQ ID NO:10). [0062]
  • FIG. 11 shows the amino acid sequence deduced from the DNA-sequence of FIG. 9 (SEQ ID NO:11). [0063]
  • FIG. 12 shows the amino acid sequence deduced from the DNA-sequence of FIG. 10 (SEQ ID NO:12). [0064]
  • FIG. 13 shows the BLASTP—alignment of 302_prot (SEQ ID NO:2) against swissnew|O94759|TRP7_HUMAN TRANSIENT RECEPTOR POTENTIIAL CHANNEL 7 (SEQ ID NO:3). [0065]
  • FIG. 14 shows the HMMPFAM—alignment of 302_prot (SEQ ID NO:2) against pfam|hmm|Trans_recep. [0066]
  • FIG. 15 shows the HMMPFAM—alignment of 302_from_mouse against pfam|hmm|MHCK_EF2_kinase MHCK/EF2 kinase domain family [0067]
  • FIG. 16 shows the genewise output[0068]
    • DETAILED DESCRIPTION OF THE INVENTION
  • The invention relates to an isolated polynucleotide from the group consisting of: [0069]
  • a) a polynucleotide encoding a transient receptor potential channel polypeptide comprising an amino acid sequence selected from the group consisting of: [0070]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 2; [0071]
  • the amino acid sequence shown in SEQ ID NO: 2; [0072]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 11; [0073]
  • the amino acid sequence shown in SEQ ID NO:11; [0074]
  • amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 12; and [0075]
  • the amino acid sequence shown in SEQ ID NO: 12. [0076]
  • b) a polynucleotide comprising the sequence of SEQ ID NOS: 1,9 or 10; [0077]
  • c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a transient receptor potential channel polypeptide; [0078]
  • d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a transient receptor potential channel polypeptide; and [0079]
  • e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a transient receptor potential channel polypeptide. [0080]
  • Furthermore, it has been discovered by the present applicant that a novel human transient receptor potential channel, particularly a human transient receptor potential channel, can be used in therapeutic methods to treat cancer, a cardiovascular disorder or a CNS disorder. Human transient receptor potential channel comprises the amino acid sequence shown in SEQ ID NO:2. A coding sequence for human transient receptor potential channel is shown in SEQ ID NO:1. This sequence is contained within the longer sequence shown in SEQ ID NO:4. This sequence is located on [0081] chromosome 9. Related ESTs (SEQ ID NOS:5-8) are expressed in kidney and retina.
  • Pfamsearch identified a TRPC family domain (PF02164) in SEQ ID NO:2. A multiple sequence alignment of SEQ ID NO:2 with other TRPCs revealed additional conserved regions. Additionally, hydrophobicity analysis predicted six membrane-spanning domains for the human transient receptor potential channel of the invention, which is a characteristic number for TRPCs. Human transient receptor potential channel is 25% identical over 896 amino acids to swissnew|O94759 (SEQ ID NO:3) (FIG. 1). [0082]
  • Human transient receptor potential channel of the invention is expected to be useful for the same purposes as previously identified transient receptor potential channels. Human transient receptor potential channel is believed to be useful in therapeutic methods to treat disorders such as cancer, cardiovascular disorders, and CNS disorders. Human transient receptor potential channel also can be used to screen for human transient receptor potential channel activators and inhibitors. [0083]
  • Polypeptides [0084]
  • Human transient receptor potential channel polypeptides according to the invention comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1133 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof, as defined below. A transient receptor potential channel polypeptide of the invention therefore can be a portion of a transient receptor potential channel protein, a full-length transient receptor potential channel protein, or a fusion protein comprising all or a portion of a transient receptor potential channel protein. [0085]
  • Biologically Active Variants [0086]
  • Human transient receptor potential channel polypeptide variants that are biologically active, e.g., retain the ability to function as an ion channel, also are transient receptor potential channel polypeptides. Preferably, naturally or non-naturally occurring transient receptor potential channel polypeptide variants have amino acid sequences which are at least about 26, 30, 35, 40, 45, 50, 55, 60, 65, or 70, preferably about 75, 80, 85, 90, 96, 96, 98, or 99% identical to the amino acid sequence shown in SEQ ID NO:2 or a fragnent thereof. Percent identity between a putative transient receptor potential channel polypeptide variant and an amino acid sequence of SEQ ID NO:2 is determined using the Blast2 alignment program (Blosum62, Expect 10, standard genetic codes). [0087]
  • Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. [0088]
  • Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity of a transient receptor potential channel polypeptide can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active transient receptor potential channel polypeptide can readily be determined by assaying for functional activity, as described for example, in the “Functional Assays” section, below. [0089]
  • Fusion Proteins [0090]
  • Fusion proteins are useful for generating antibodies against transient receptor potential channel polypeptide amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins that interact with portions of a transient receptor potential channel polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens. [0091]
  • A transient receptor potential channel polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1133 contiguous amino acids of SEQ ID NO:2 or of a biologically active variant, such as those described above. The first polypeptide segment also can comprise full-length transient receptor potential channel protein. [0092]
  • The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include β-galactosidase, β-glucuonidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the transient receptor potential channel polypeptide-encoding sequence and the heterologous protein sequence, so that the transient receptor potential channel polypeptide can be cleaved and purified away from the heterologous moiety. [0093]
  • A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO:1 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, Wis.), Stratagene (La Jolla, Calif.), CLONTECH (Mountan View, Calif.), Santa Cruz Biotechnology (Santa Cruz, Calif.), MBL International Corporation (MIC; Watertown, Mass.), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS). [0094]
  • Identification of Species Homologs [0095]
  • Species homologs of human transient receptor potential channel polypeptide can be obtained using transient receptor potential channel polypeptide polynucleotides (described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifyig cDNAs which encode homologs of transient receptor potential channel polypeptide, and expressing the cDNAs as is known in the art. [0096]
  • Polynucleotides [0097]
  • A transient receptor potential channel polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a transient receptor potential channel polypeptide. A coding sequence for human transient receptor potential channel is shown in SEQ ID NO:1. [0098]
  • Degenerate nucleotide sequences encoding human transient receptor potential channel polypeptides, as well as homologous nucleotide sequences which are at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in SEQ ID NO:1 or its complement also are transient receptor potential channel polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of −12 and a gap extension penalty of −2. Complementary DNA (cDNA) molecules, species homologs, and variants of transient receptor potential channel polynucleotides that encode biologically active transient receptor potential channel polypeptides also are transient receptor potential channel polynucleotides. Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO:1 or its complement also are transient receptor potential channel polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides. [0099]
  • Identification of Polynucleotide Variants and Homologs [0100]
  • Variants and homologs of the transient receptor potential channel polynucleotides described above also are transient receptor potential channel polynucleotides. Typically, homologous transient receptor potential channel polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known transient receptor potential channel polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions—2×SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2×SSC, 0.1% SDS, 50° C. once, 30 minutes; then 2×SSC, room temperature twice, 10 minutes each-homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. [0101]
  • Species homologs of the transient receptor potential channel polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of transient receptor potential channel polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T[0102] m of a double-stranded DNA decreases by 1-1.5° C. with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Variants of human transient receptor potential channel polynucleotides or transient receptor potential channel polynucleotides of other species can therefore be identified by hybridizing a putative homologous transient receptor potential channel polynucleo-tide with a polynucleotide having a nucleotide sequence of SEQ ID NO:1 or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.
  • Nucleotide sequences which hybridize to transient receptor potential channel poly-nucleotides or their complements following stringent hybridization and/or wash conditions also are transient receptor potential channel polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51. [0103]
  • Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20° C. below the calculated T[0104] m of the hybrid under study. The Tm of a hybrid between a transient receptor potential channel polynucleotide having a nucleotide sequence shown in SEQ ID NO:1 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98% identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):
  • Tm=81.5° C. ˜16.6(log10[Na+])+0.41(%G+C)−0.63(%formamide)−600/l),
  • where l=the length of the hybrid in basepairs. [0105]
  • Stringent wash conditions include, for example, 4×SSC at 65° C., or 50% formamide, 4×SSC at 42° C., or 0.5×SSC, 0.1% SDS at 65° C. Highly stringent wash conditions include, for example, 0.2×SSC at 65° C. [0106]
  • Preparation of Polynucleotides [0107]
  • A transient receptor potential channel polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Poly-nucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated transient receptor potential channel polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise transient receptor potential channel nucleotide sequences. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules. [0108]
  • Human transient receptor potential channel cDNA molecules can be made with standard molecular biology techniques, using transient receptor potential channel mRNA as a template. Human transient receptor potential channel cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template. [0109]
  • Alternatively, synthetic chemistry techniques can be used to synthesize transient receptor potential channel polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a transient receptor potential channel polypeptide having, for example, an amino acid sequence shown in SEQ ID NO:2 or a biologically active variant thereof. [0110]
  • Extending Polynucleotides [0111]
  • The partial sequence disclosed herein can be used to identify the corresponding full length gene from which it was derived. The partial sequence can be nick-translated or end-labeled with [0112] 32P using polynucleotide kinase using labeling methods known to those with skill in the art (BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al., eds., Elsevier Press, N.Y., 1986). A lambda library prepared from human tissue can be directly screened with the labeled sequences of interest or the library can be converted en masse to pBluescript (Stratagene Cloning Systems, La Jolla, Calif. 92037) to facilitate bacterial colony screening (see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press (1989, pg. 1.20).
  • Both methods are well known in the art. Briefly, filters with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured, and the DNA is fixed to the filters. The filters are hybridized with the labeled probe using hybridization conditions described by Davis et al., 1986. The partial sequences, cloned into lambda or pBluescript, can be used as positive controls to assess background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification. The resulting autoradio-grams are compared to duplicate plates of colonies or plaques; each exposed spot corresponds to a positive colony or plaque. The colonies or plaques are selected, expanded and the DNA is isolated from the colonies for further analysis and sequencing. [0113]
  • Positive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial sequence and the other primer from the vector. Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysis. [0114]
  • Once one or more overlapping cDNA clones are identified, the complete sequence of the clones can be determined, for example after exonuclease III digestion (McCombie et al., [0115] Methods 3, 33-40, 1991). A series of deletion clones are generated, each of which is sequenced. The resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence.
  • Various PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR [0116] Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al., [0117] Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72° C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al., [0118] PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.
  • Another method which can be used to retrieve unknown sequences is that of Parker et al., [0119] Nucleic Acids Res. 19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • When screening for fiull-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5′ regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5′ non-transcribed regulatory regions. [0120]
  • Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample. [0121]
  • Obtaining Polypeptides [0122]
  • Human transient receptor potential channel polypeptides can be obtained, for example, by purification from human cells, by expression of transient receptor potential channel polynucleotides, or by direct chemical synthesis. [0123]
  • Protein Purification [0124]
  • Human transient receptor potential channel polypeptides can be purified from any cell that expresses the polypeptide, including host cells that have been transfected with transient receptor potential channel expression constructs. A purified transient receptor potential channel polypeptide is separated from other compounds that normally associate with the transient receptor potential channel polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A preparation of purified transient receptor potential channel polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. [0125]
  • Expression of Polynucleotides [0126]
  • To express a transient receptor potential channel polynucleotide, the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding transient receptor potential channel polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989. [0127]
  • A variety of expression vector/host systems can be utilized to contain and express sequences encoding a transient receptor potential channel polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems. [0128]
  • The control elements or regulatory sequences are those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagernid (Stratagene, LaJolla, Calif.) or pSPORT1 plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a transient receptor potential channel polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker. [0129]
  • Bacterial and Yeast Expression Systems [0130]
  • In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the transient receptor potential channel polypeptide. For example, when a large quantity of a transient receptor potential channel polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional [0131] E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding the transient receptor potential channel polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced. pIN vectors (Van Heeke & Schuster, J BioL Chem. 264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • In the yeast [0132] Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) and Grant et al., Methods Enzymol. 153, 516-544, 1987.
  • Plant and Insect Expression Systems [0133]
  • If plant expression vectors are used, the expression of sequences encoding transient receptor potential channel polypeptides can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, [0134] EMBO J. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al., EMBO J. 3, 1671-1680, 1984; Broglie et al., Science 224, 838-843, 1984; Winter et al., Results Probl. Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).
  • An insect system also can be used to express a transient receptor potential channel polypeptide. For example, in one such system [0135] Autographa californica nuclear polyhedrosis virus (ACNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding transient receptor potential channel polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of transient receptor potential channel polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which transient receptor potential channel polypeptides can be expressed (Engelhard et al., Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).
  • Mammalian Expression Systems [0136]
  • A number of viral-based expression systems can be used to express transient receptor potential channel polypeptides in mammalian host cells. For example, if an adeno-virus is used as an expression vector, sequences encoding transient receptor potential channel polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing a transient receptor potential channel polypeptide in infected host cells (Logan & Shenk, [0137] Proc. Nat. Acad. Sci. 81, 3655-3659, 1984). If desired, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles). [0138]
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding transient receptor potential channel polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a transient receptor potential channel polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al., [0139] Results Probl. Cell Differ. 20, 125-162, 1994).
  • Host Cells [0140]
  • A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed transient receptor potential channel polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro”form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein. [0141]
  • Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express transient receptor potential channel polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced transient receptor potential channel sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986. [0142]
  • Any number of selection systems can be used to recover-transformed cell lines. [0143]
  • These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., [0144] Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al., Cell 22, 817 -23, 1980) genes which can be employed in tk or aprf cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77, 3567-70, 1980), npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al., Methods Mol. Biol. 55, 121-131, 1995).
  • Detecting Expression [0145]
  • Although the presence of marker gene expression suggests that the transient receptor potential channel polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a transient receptor potential channel polypeptide is inserted within a marker gene sequence, transformed cells containing sequences that encode a transient receptor potential channel polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a transient receptor potential channel polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the transient receptor potential channel polynucleotide. [0146]
  • Alternatively, host cells which contain a transient receptor potential channel polynucleotide and which express a transient receptor potential channel polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding a transient receptor potential channel polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleo-tides encoding a transient receptor potential channel polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a transient receptor potential channel polypeptide to detect transformants that contain a transient receptor potential channel polynucleotide. [0147]
  • A variety of protocols for detecting and measuring the expression of a transient receptor potential channel polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immuno-assay using monoclonal antibodies reactive to two non-interfering epitopes on a transient receptor potential channel polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al., SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al., [0148] J. Exp. Med. 158, 1211-1216, 1983).
  • A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding transient receptor potential channel polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a transient receptor potential channel polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like. [0149]
  • Expression and Purification of Polypeptides [0150]
  • Host cells transformed with nucleotide sequences encoding a transient receptor potential channel polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode transient receptor potential channel polypeptides can be designed to contain signal sequences which direct secretion of soluble transient receptor potential channel polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound transient receptor potential channel polypeptide. [0151]
  • As discussed above, other constructions can be used to join a sequence encoding a transient receptor potential channel polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and the transient receptor potential channel polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a transient receptor potential channel polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al., [0152] Prot. Exp. Purif 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the transient receptor potential channel polypeptide from the fusion protein. Vectors that contain fusion proteins are disclosed in Kroll et al., DNA Cell Bio. 12, 441-453, 1993.
  • Chemical Synthesis [0153]
  • Sequences encoding a transient receptor potential channel polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al., [0154] Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980). Alternatively, a transient receptor potential channel polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al., Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of transient receptor potential channel polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic transient receptor potential channel polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the transient receptor potential channel polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fuision protein. [0155]
  • Production of Altered Polypeptides [0156]
  • As will be understood by those of skill in the art, it may be advantageous to produce transient receptor potential channel polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life that is longer than that of a transcript generated from the naturally occurring sequence. [0157]
  • The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter transient receptor potential channel polypeptide-encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth. [0158]
  • Antibodies [0159]
  • Any type of antibody known in the art can be generated to bind specifically to an epitope of a transient receptor potential channel polypeptide. “Antibody” as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab′)[0160] 2, and Fv, which are capable of binding an epitope of a transient receptor potential channel polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.
  • An antibody which specifically binds to an epitope of a transient receptor potential channel polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen. [0161]
  • Typically, an antibody which specifically binds to a transient receptor potential channel polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to transient receptor potential channel polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a transient receptor potential channel polypeptide from solution. [0162]
  • Human transient receptor potential channel polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a transient receptor potential channel polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG ([0163] bacilli Calmette-Guerin) and Corynebacterium parvum are especially useful.
  • Monoclonal antibodies that specifically bind to a transient receptor potential channel polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoina technique (Kohler et al., [0164] Nature 256, 495-497, 1985; Kozbor et al., J. Immunol. Methods 81, 31-42, 1985; Cote et al., Proc. Natl. Acad. Sci. 80,2026-2030, 1983; Cole et al., Mol. Cell Biol. 62, 109-120, 1984).
  • In addition, techniques developed for the production of “chimeric antibodies,” the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used Morrison et al., [0165] Proc. Natl. Acad. Sci. 81, 6851-6855, 1984; Neuberger et al., Nature 312, 604-608, 1984; Takeda et al., Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be “humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies that specifically bind to a transient receptor potential channel polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. Pat. No. 5,565,332.
  • Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to transient receptor potential channel polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, [0166] Proc. Natl. Acad. Sci. 88, 11120-23, 1991).
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al., 1996, [0167] Eur. J Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.
  • A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al., 1995, [0168] Int. J. Cancer 61, 497-501; Nicholls et al., 1993, J. Immunol. Meth. 165, 81-91).
  • Antibodies which specifically bind to transient receptor potential channel poly-peptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al., [0169] Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al., Nature 349, 293-299, 1991).
  • Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the “diabodies” described in WO 94/13804, also can be prepared. [0170]
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a transient receptor potential channel polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration. [0171]
  • Antisense Oligonucleotides [0172]
  • Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of transient receptor potential channel gene products in the cell. [0173]
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5′ end of one nucleotide with the 3′ end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, [0174] Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al., Chem. Rev. 90, 543-583,1990.
  • Modifications of transient receptor potential channel gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5′, or regulatory regions of the transient receptor potential channel gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions −10and +10 from the start site, are preferred. Similarly, inhibition can be achieved using “triple helix” base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al., in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. [0175]
  • Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a transient receptor potential channel polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a transient receptor potential channel polynucleotide, each separated by a stretch of contiguous nucleotides which are not. complementary to adjacent transient receptor potential channel nucleotides, can provide sufficient targeting specificity for transient receptor potential channel mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular transient receptor potential channel polynucleotide sequence. [0176]
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a transient receptor potential channel polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, intemucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3′, 5′-substituted oligonucleotide in which the 3′ hydroxyl group or the 5′ phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al., [0177] Trends Biotechnol. 10, 152-158, 1992; Uhlmann et al., Chem. Rev. 90, 543-584, 1990; Uhlmann et al., Tetrahedron Lett. 215, 3539-3542, 1987.
  • Ribozymes [0178]
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, [0179] Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Pat. No. 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hanmnerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • The coding sequence of a transient receptor potential channel polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the transient receptor potential channel polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. [0180] Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete “hybridization” region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al., EP 321,201).
  • Specific ribozyme cleavage sites within a transient receptor potential channel RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate transient receptor potential channel RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target. [0181]
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease transient receptor potential channel expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells. [0182]
  • As taught in Haseloff et al., U.S. Pat. No. 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells. [0183]
  • Differentially Expressed Genes [0184]
  • Described herein are methods for the identification of genes whose products interact with human transient receptor potential channel. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, cancer, cardiovascular disorders, and CNS disorders. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human transient receptor potential channel gene or gene product may itself be tested for differential expression. [0185]
  • The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis. [0186]
  • Identification of Differentially Expressed Genes [0187]
  • To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al., ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Pat. No. 4,843,155. [0188]
  • Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al., [0189] Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al., Nature 308, 149-53; Lee et al., Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and, preferably, differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S. Pat. No. 5,262,311).
  • The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human transient receptor potential channel. For example, treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human transient receptor potential channel. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human transient receptor potential channel gene or gene product are up-regulated or down-regulated. [0190]
  • Screening Methods [0191]
  • The invention provides assays for screening test compounds that bind to or modulate the activity of a transient receptor potential channel polypeptide or a transient receptor potential channel polynucleotide. A test compound preferably binds to a transient receptor potential channel polypeptide or polynucleotide. More preferably, a test compound decreases or increases functional activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound. [0192]
  • Test Compounds [0193]
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinately or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the “one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, [0194] Anticancer Drug Des. 12, 145, 1997.
  • Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al., [0195] Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J Med Chem 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994; Carell et al., Angew. Chem Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al., Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirla et al., Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J. Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Pat. No. 5,223,409).
  • High Throughput Screening [0196]
  • Test compounds can be screened for the ability to bind to transient receptor potential channel polypeptides or polynucleotides or to affect transient receptor potential channel activity or transient receptor potential channel gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format. [0197]
  • Alternatively, “free format assays,” or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al., [0198] Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.
  • Another example of a free format assay is described by Chelsky, “Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches,” reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-[0199] 10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.
  • Yet another example is described by Salmon et al., [0200] Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.
  • Another high throughput screening method is described in Beutel et al., U.S. Pat. No. 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together. [0201]
  • Binding Assays [0202]
  • For binding assays, the test compound is preferably a small molecule that binds to the transient receptor potential channel polypeptide such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules. [0203]
  • In binding assays, either the test compound or the transient receptor potential channel polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound that is bound to the transient receptor potential channel polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product. [0204]
  • Alternatively, binding of a test compound to a transient receptor potential channel polypeptide can be determined without labeling either of the interactants. For example , a microphysiometer can be used to detect binding of a test compound with a transient receptor potential channel polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a transient receptor potential channel polypeptide (McConnell et al., [0205] Science 257, 1906-1912, 1992).
  • Determining the ability of a test compound to bind to a transient receptor potential channel polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, [0206] Anal. Chem. 63, 2338-2345, 1991, and Szabo et al., Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • In yet another aspect of the invention, a transient receptor potential channel polypeptide can be used as a “bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., [0207] Cell 72, 223-232, 1993; Madura et al., J Biol. Chem. 268, 12046-12054, 1993; Bartel et al., BioTechniques 14, 920-924, 1993; Iwabuchi et al., Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the transient receptor potential channel polypeptide and modulate its activity.
  • The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding a transient receptor potential channel polypeptide can be fused to a poly-nucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein (“prey” or “sample”) can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transciptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the transient receptor potential channel polypeptide. [0208]
  • It may be desirable to immobilize either the transient receptor potential channel poly-nucleotide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the transient receptor potential channel polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a transient receptor potential channel polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. [0209]
  • In one embodiment, the transient receptor potential channel polypeptide is a fusion protein comprising a domain that allows the transient receptor potential channel polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed transient receptor potential channel polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined. [0210]
  • Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a transient receptor potential channel polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated transient receptor potential channel polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to a transient receptor potential channel poly-peptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation. [0211]
  • Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the transient receptor potential channel polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the transient receptor potential channel polypeptide, and SDS gel electrophoresis under non-reducing conditions. [0212]
  • Screening for test compounds which bind to a transient receptor potential channel polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a transient receptor potential channel polypeptide or polynucleotide can be used in a cell-based assay system. A transient receptor potential channel poly-nucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a transient receptor potential channel polypeptide or polynucleotide is determined as described above. [0213]
  • Functional Assays [0214]
  • Test compounds can be tested for the ability to increase or decrease a biological effect of a human transient receptor potential channel. Such biological effects can be determined for example using functional assays such as those described below. Functional assays can be carried out after contacting either a purified transient receptor potential channel polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound which increases or decreases a functional activity of a transient receptor potential channel polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent. [0215]
  • Ion channels can be tested functionally in living cells. Polypeptides comprising amino acid sequences encoded by open reading frames of the invention are either expressed endogeneously in appropriate reporter cells or are introduced recombinantly. Channel activity can be monitored by concentration changes of the permeating ion, by changes in the transmembrane electrical potential gradient, or by measuring a cellular response (e.g., expression of a reporter gene or secretion of a neuro-transmitter triggered or modulated by the polypeptide's activity. [0216]
  • The activity of ion channel proteins in cells can be determined, for example, by loading the cells with an ion-sensitive fluorescent indicator. Fluorescent indicators can be loaded into cells in 96-well plates or another container, and the activity of ion channel proteins in the presence or absence of various test compounds can be simply and rapidly determined. See, e.g., U.S. Pat. No. 6,057,114. Ion channel currents result in changes of electrical membrane potential (V[0217] m) which can be monitored directly using potentiometric fluorescent probes. These electrically charged indicators (e.g., the anionic oxonol dye DiBAC4(3)) redistribute between extra-and intracellular compartments in response to voltage changes across the membrane in which the ion channel resides. The equilibrium distribution is governed by the Nemst-equation. Thus, changes in membrane potential results in concomitant changes in cellular fluorescence. Again, changes in Vm might be caused directly by the activity of the target ion channel or through amplification and/or prolongation of the signal by channels co-expressed in the same cell.
  • Another approach to determining the activity of ion channel proteins involves the electrophysiological determination of ionic currents. Cells which endogenously express a transient receptor potential channel can be used to study the effects of various test compounds or transient receptor potential channel polypeptides on endogenous ionic currents attributable to the activity of transient receptor potential channels. Alternatively, cells which do not express transient receptor potential channel can be employed as hosts for the expression of transient receptor potential channel, whose activity can then be studied by electrophysiological or other means. Cells preferred as host cells for the heterologous expression of transient receptor potential channel are preferably mammalian cells such as COS cells, mouse L cells, CHO cells (e.g., DG44 cells), human embryonic kidney cells (e.g., HEK293 cells), African green monkey cells and the like; amphibian cells, such as [0218] Xenopus laevis oocytes; or cells of yeast such as S. cerevisiae or P. pastoris. See, e.g., U.S. Pat. No. 5,876,958.
  • Electrophysiological procedures for measuring the current across a cell membrane are well known. A preferred method is the use of a voltage clamp as in the whole-cell patch clamp technique. Non-calcium currents can be eliminated by established methods so as to isolate the ionic current flowing through ion channel proteins. In the case of heterologously expressed transient receptor potential channel, ionic currents resulting from endogenous ion channel proteins can be suppressed by known pharmacological or electrophysiological techniques. See, e.g., U.S. Pat. No. 5,876,958. [0219]
  • A further activity of the transient receptor potential channel which can be assessed is its ability to bind various ligands, including test compounds. The ability of a test compound to bind transient receptor potential channel or fragments thereof may be determined by any appropriate competitive binding analysis (e.g., Scatchard plots), wherein the binding capacity and/or affinity is determined in the presence and absence of one or more concentrations a compound having known affinity for the transient receptor potential channel. Binding assays can be performed using whole cells that express transient receptor potential channel (either endogenously or heterologously), membranes prepared from such cells, or purified transient receptor potential channel. [0220]
  • Gene Expression [0221]
  • In another embodiment, test compounds that increase or decrease transient receptor potential channel gene expression are identified. A transient receptor potential channel polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the transient receptor potential channel polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression. [0222]
  • The level of transient receptor potential channel mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a transient receptor potential channel polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a transient receptor potential channel polypeptide. [0223]
  • Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell that expresses a transient receptor potential channel polynucleotide can be used in a cell-based assay system. The transient receptor potential channel polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used. [0224]
  • Pharmaceutical Compositions [0225]
  • The invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, a transient receptor potential channel polypeptide, transient receptor potential channel polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a transient receptor potential channel polypeptide, or mimetics, activators, or inhibitors of a transient receptor potential channel polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones. [0226]
  • In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. [0227]
  • Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. [0228]
  • Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage. [0229]
  • Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers. [0230]
  • Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. [0231]
  • The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1% -2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use. [0232]
  • Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON's PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration. [0233]
  • Therapeutic Indications and Methods [0234]
  • Human transient receptor potential channel (TRPC) genes share sequence similarity with Drosophila transient receptor potential gene. Recent studies found expression of TRPC homologues in vascular endothelium, indicating that TRP cation channels from the molecular basis of endothelial oxidant-activated cation channels in endothelial cells (7). In invertebrates, TRPCs are expressed principally in the eye. In vertebrates, however, TRPCs have a wide tissue distribution, including CNS and cardiovascular tissues. Recently, the MTR1 gene, which shows highest homology to the human TRPC7 and melastatin genes, was identified and characterized (6). MTR1 is expressed in many rhabdomyosarcomas and Wilm's tumors, suggesting a role of TRPCs in tumor growth (6). Mutations in TRPCs are found in patients who have mucolipidosis type IV, which is a neurodegenerative disorder. Thus, it is believed that the human transient receptor potential channel of the invention can be regulated to treat cancer, cardiovascular disorders, and CNS disorders. [0235]
  • Cancer. [0236]
  • Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recruit blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Various combinations of these aberrant physiologies, along with the acquisition of drug-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue. [0237]
  • Most standard cancer therapies target cellular proliferation and rely on the differential proliferative capacities between transformed and normal cells for their efficacy. This approach is hindered by the facts that several important normal cell types are also highly proliferative and that cancer cells frequently become resistant to these agents. Thus, the therapeutic indices for traditional anti-cancer therapies rarely exceed 2.0. [0238]
  • The advent of genomics-driven molecular target identification has opened up the possibility of identifying new cancer-specific targets for therapeutic intervention that will provide safer, more effective treatments for cancer patients. Thus, newly discovered tumor-associated genes and their products can be tested for their role(s) in disease and used as tools to discover and develop innovative therapies. Genes playing important roles in any of the physiological processes outlined above can be characterized as cancer targets. [0239]
  • Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins. These proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities. Activators and/or inhibitors of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drug development and subsequent testing in humans. [0240]
  • Cardiovascular Disorders [0241]
  • Cardiovascular diseases include the following disorders of the heart and the vascular system: congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, and peripheral vascular diseases. [0242]
  • Heart failure is defined as a pathophysiologic state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failure, such as high-output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause. [0243]
  • Myocardial infarction (MI) is generally caused by an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously narrowed by arteriosclerosis. MI prophylaxis (primary and secondary prevention) is included, as well as the acute treatment of MI and the prevention of complications. [0244]
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen. This group of diseases includes stable angina, unstable angina, and asymptomatic ischemia. [0245]
  • Arrhythmias include all forms of atrial and ventricular tachyarrhythmias (atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexcitation syndrome, ventricular tachycardia, ventricular flutter, and ventricular fibrillation), as well as bradycardic forms of arrhythmias. [0246]
  • Vascular diseases include primary as well as all kinds of secondary arterial hypertension (renal, endocrine, neurogenic, others). The disclosed gene and its product may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications. [0247]
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon, and venous disorders. [0248]
  • CNS Disorders [0249]
  • Central and peripheral nervous system disorders also can be treated, such as primary and secondary disorders after brain injury, disorders of mood, anxiety disorders, disorders of thought and volition, disorders of sleep and wakefulness, diseases of the motor unit, such as neurogenic and myopathic disorders, neurodegenerative disorders such as Alzheimer's and Parkinson's disease, and processes of peripheral and chronic pain. [0250]
  • Pain that is associated with CNS disorders also can be treated by regulating the activity of human transient receptor potential channel. Pain which can be treated includes that associated with central nervous system disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemorrhage, vascular malformation). Non-central neuropathic pain includes that associated with post mastectomy pain, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-herpetic neuralgia. Pain associated with cancer and cancer treatment also can be treated, as can headache pain (for example, migraine with aura, migraine without aura, and other migraine disorders), episodic and chronic tension-type headache, tension-type like headache, cluster headache, and chronic paroxysmal hemicrania. [0251]
  • This invention flier pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a transient receptor potential channel polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. [0252]
  • A reagent which affects transient receptor potential channel activity can be administered to a human cell, either in vitro or in vivo, to reduce transient receptor potential channel activity. The reagent preferably binds to an expression product of a human transient receptor potential channel gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art. [0253]
  • In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin. [0254]
  • A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma mernbrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about 0.5 μg of DNA per 16 nmole of liposome delivered to about 10[0255] 6 cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 106 cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 106 cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
  • Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome. [0256]
  • Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Pat. No. 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes. [0257]
  • In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. [0258] Trends in Biotechnol. 11, 202-05 (1993); Chiou et al., GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al., J. Biol. Chem. 269, 542-46 (1994); Zenke et al., Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al., J. Biol. Chem. 266, 338-42 (1991).
  • Determination of a Therapeutically Effective Dose [0259]
  • The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases transient receptor potential channel activity relative to the transient receptor potential channel activity which occurs in the absence of the therapeutically effective dose. [0260]
  • For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. [0261]
  • Therapeutic efficacy and toxicity, e.g., ED[0262] 50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED[0263] 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation. [0264]
  • Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. [0265]
  • If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, “gene gun,” and DEAE- or calcium phosphate-mediated transfection. [0266]
  • Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA. [0267]
  • If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above. [0268]
  • Preferably, a reagent reduces expression of a transient receptor potential channel gene or the activity of a transient receptor potential channel polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a transient receptor potential channel gene or the activity of a transient receptor potential channel polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to transient receptor potential channel-specific mRNA, quantitative RT-PCR, immunologic detection of a transient receptor potential channel polypeptide, or measurement of transient receptor potential channel activity. [0269]
  • In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatmnent or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. [0270]
  • Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. [0271]
  • Diagnostic Methods [0272]
  • Human transient receptor potential channel also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the polypeptide. For example, differences can be determined between the cDNA or genomic sequence encoding transient receptor potential channel in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease. [0273]
  • Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags. [0274]
  • Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., [0275] Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci. U.S.A 85, 4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
  • Altered levels of transient receptor potential channel also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays. [0276]
  • All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention. [0277]
    • EXAMPLE 1
  • Detection of Transient Receptor Potential Channel Activity [0278]
  • The polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4-transient receptor potential channel polypeptide obtained is transfected into human embryonic kidney 293 cells. 2 days after transfection, cells attached to coverslip are washed twice with HPSS 8120 ml mM NaCl, 5.3 mM KC1, 0.8 mM CaC12, 11.1 mM glucose, 20 mM Hepes (pH 7.4) and loaded with fura 2-AM (Molecular Probes, 5μm in HPSS) for 30 minutes at room temperature in the dark. After washing and incubating in fresh HPSS for 30 min at room temperature to achieve de-esterification, the coverslips are inserted into a cicular open-bottom chamber and placed onto the stage of a Zeiss Axovert microscope fitted with an Attofluor Digital Imaging and Photometry System(Attofluor Inc., Rockville, Md.) 20-30 isolated fura 2-loaded cells are selected and [Ca2+] i in these cells is measured by1 fluorescence videomicroscopy at room temperature using alternating excitation wavelengths of 334 and 380 nm and monitoring emitted fliorescence at 520 nm. Free [Ca2+] i is calculated from 334/380 fluorescence ratios following the method described previously. All reagents are diluted to their final concentrations in HPSS and applied to the cells by surface perfusion. It is shown that the polypeptide of SEQ ID NO: 2 has a transient receptor potential channel activity. [0279]
    • EXAMPLE 2
  • Expression of Recombinant Human Transient Receptor Potential Channel [0280]
  • The [0281] Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, Calif.) is used to produce large quantities of recombinant human transient receptor potential channel polypeptides in yeast. The transient receptor potential channel-encoding DNA sequence is derived from SEQ ID NO:1. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5′-end an initiation codon and at its 3′-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.
  • The yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, Calif.) according to manufacturer's instructions. Purified human transient receptor potential channel polypeptide is obtained. [0282]
    • EXAMPLE 3
  • Identification of Test Compounds that Bind to Transient Receptor Potential Channel Polypeptides [0283]
  • Purified transient receptor potential channel polypeptides comprising a glutathione-S-transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Human transient receptor potential channel polypeptides comprise the amino acid sequence shown in SEQ ID NO:2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound [0284]
  • The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a transient receptor potential channel polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a transient receptor potential channel polypeptide. [0285]
    • EXAMPLE 4
  • Identification of a Test Compound Which Decreases Transient Receptor Potential Channel Gene Expression [0286]
  • A test compound is administered to a culture of human cells transfected with a transient receptor potential channel expression construct and incubated at 37° C. for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control. [0287]
  • RNA is isolated from the two cultures as described in Chirgwin et al., [0288] Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a 32P-labeled transient receptor potential channel-specific probe at 65° C. in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO:1. A test compound that decreases the transient receptor potential channel-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of transient receptor potential channel gene expression.
    • EXAMPLE 5
  • Tissue-Specific Expression of Transient Receptor Potential Channel [0289]
  • The qualitative expression pattern of transient receptor potential channel in various tissues is determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). [0290]
  • To demonstrate that transient receptor potential channel is involved in CNS disorders, the following tissues are screened: fetal and adult brain, muscle, heart, lung, kidney, liver, thymus, testis, colon, placenta, trachea, pancreas, kidney, gastric mucosa, colon, liver, cerebellum, skin, cortex (Alzheimer's and normal), hypothalamus, cortex, amygdala, cerebellum, hippocampus, choroid, plexus, thalamus, and spinal cord. [0291]
  • To demonstrate that transient receptor potential channel is involved in cancer, expression is determined in the following tissues: adrenal gland, bone marrow, brain, cerebellum, colon, fetal brain, fetal liver, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, uterus, and peripheral blood lymphocytes. Expression in the following cancer cell lines also is determined: DU-145 (prostate), NCI-H125 (lung), HT-29 (colon), COLO-205 (colon), A-549 (lung), NCI-H460 (lung), HT-116 (colon), DLD-1 (colon), MDA-MD-231 (breast), LS174T (colon), ZF-75 (breast), MDA-MN-435 (breast), HT-1080, MCF-7 (breast), and U87. Matched pairs of malignant and normal tissue from the same patient also are tested. [0292]
  • Quantitative expression profiling. Quantitative expression profiling is performed by the form of quantitative PCR analysis called “kinetic analysis” firstly described in Higuchi et al., [0293] BioTechnology 10, 413-17, 1992, and Higuchi et al., BioTechnology 11, 1026-30, 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies.
  • If the amplification is performed in the presence of an internally quenched fluorescent oligonucleotide (TaqMan probe) complementary to the target sequence, the probe is cleaved by the 5′-3′ endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al., [0294] Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al., Genome Res. 6, 986-94, 1996, and Gibson et al., Genome Res. 6, 995-1001, 1996).
  • The amplification of an endogenous control can be performed to standardize the amount of sample RNA added to a reaction. In this kind of experiment, the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous control can be independently quantified in the same tube if probes labeled with different dyes are used. [0295]
  • All “real time PCR” measurements of fluorescence are made in the ABI Prism 7700. [0296]
  • RNA extraction and cDNA preparation. Total RNA from the tissues listed above are used for expression quantification. RNAs labeled “from autopsy” were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, Md.) according to the manufacturer's protocol. [0297]
  • Fifty μg of each RNA were treated with DNase I for 1 hour at 37 C in the following reaction mix: 0.2 U/μl RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/μl RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; 10mM MgCl[0298] 2; 50 mM NaCl; and 1 mM DTT.
  • After incubation, RNA is extracted once with 1 volume of phenol:chloroform:isoamyl alcohol (24:24:1) and once with chloroform, and precipitated with {fraction (1/10)} volume of 3 M NaAcetate, pH5.2, and 2 volumes of ethanol. [0299]
  • Fifty μg of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, Tex.). After resuspension and spectrophotometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200 ng/μL. Reverse transcription is carried out with 2.5μM of random hexamer primers. [0300]
  • TaqMan quantitative analysis. Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5′ end FAM (6-carboxy-fluorescein) and at the 3′ end with TAMRA (6-carboxy-tetramethyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate. [0301]
  • Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA). [0302]
  • The assay reaction mix is as follows: 1× final TaqMan Universal PCR Master Mix (from 2× stock) (PE Applied Biosystems, CA); 1× PDAR control—18S RNA (from 20× stock); 300 nM forward primer; 900 nM reverse primer; 200 nM probe; 10 ng cDNA; and water to 25 μl. [0303]
  • Each of the following steps are carried out once: pre PCR, 2 minutes at 50° C., and 10 minutes at 95° C. The following steps are carried out 40 times: denaturation, 15 seconds at 95° C., annealing/extension, 1 minute at 60° C. [0304]
  • The experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA). At the end of the run, fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity. [0305]
    • EXAMPLE 6
  • In vivo Testing of Compounds/target Validation [0306]
  • 1. Acute Mechanistic Assays [0307]
  • 1.1. Reduction in Mitogenic Plasma Hormone Levels [0308]
  • This non-tumor assay measures the ability of a compound to reduce either the endogenous level of a circulating hormone or the level of hormone produced in response to a biologic stimulus. Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.). At a predetermined time after administration of test compound, blood plasma is collected. Plasma is assayed for levels of the hormone of interest. If the normal circulating levels of the hormone are too low and/or variable to provide consistent results, the level of the hormone may be elevated by a pre-treatment with a biologic stimulus (i.e., LHRH may be injected i.m. into mice at a dosage of 30 ng/mouse to induce a burst of testosterone synthesis). The timing of plasma collection would be adjusted to coincide with the peak of the induced hormone response. Compound effects are compared to a vehicle-treated control group. An F-test test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value≦0.05 compared to the vehicle control group. [0309]
  • 1.2. Hollow Fiber Mechanism of Action Assay [0310]
  • Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol, these may include assays for gene expression (bDNA, PCR, or Taqman), or a specific biochemical activity (i.e., cAMP levels. Results are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p≦0.05 as compared to the vehicle control group. [0311]
  • 2. Subacute Functional In Vivo Assays [0312]
  • 2.1. Reducton in Mass of Hormone Dependent Tissues [0313]
  • This is another non-tumor assay that measures the ability of a compound to reduce the mass of a hormone dependent tissue (i.e., seminal vesicles in males and uteri in females). Rodents are administered test compound (p.o., i.p., i.v., i.m., or s.c.) according to a predetermined schedule and for a predetermined duration (i.e., 1 week). At termination of the study, animals are weighed, the target organ is excised, any fluid is expressed, and the weight of the organ is recorded. Blood plasma may also be collected. Plasma may be assayed for levels of a hormone of interest or for levels of test agent. Organ weights may be directly compared or they may be normalized for the body weight of the animal. Compound effects are compared to a vehicle-treated control group. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test. Significance is p value≦0.05 compared to the vehicle control group. [0314]
  • 2.2. Hollow Fiber Proliferation Assay [0315]
  • Hollow fibers are prepared with desired cell line(s) and implanted intraperitoneally and/or subcutaneously in rodents. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Fibers are harvested in accordance with specific readout assay protocol. Cell proliferation is determined by measuring a marker of cell number (i.e., MTT or LDH). The cell number and change in cell number from the starting inoculum are analyzed by Student's t-test or Rank Sum test after the variance between groups is compared by an F-test, with significance at p≦0.05 as compared to the vehicle control group. [0316]
  • 2.3. Anti-angiogenesis Models [0317]
  • 2.3.1. Corneal Angiogenesis [0318]
  • Hydron pellets with or without growth factors or cells are implanted into a micropocket surgically created in the rodent cornea. Compound administration may be systemic or local (compound mixed with growth factors in the hydron pellet). Corneas are harvested at 7 days post implantation immediately following intracardiac infusion of colloidal carbon and are fixed in 10% formalin. Readout is qualitative scoring and/or image analysis. Qualitative scores are compared by Rank Sum test. Image analysis data is evaluated by measuring the area of neovascularization (in pixels) and group averages are compared by Student's t-test (2 tail). Significance is p ≦0.05 as compared to the growth factor or cells only group. [0319]
  • 2.3.2. Mairigel Angiogenesis [0320]
  • Matrigel, containing cells or growth factors, is injected subcutaneously. Compounds are administered p.o., i.p., i.v., i.m., or s.c. Matrigel plugs are harvested at predetermined time point(s) and prepared for readout. Readout is an ELISA-based assay for hemoglobin concentration and/or histological examination (i.e. vessel count, special staining for endothelial surface markers: CD31, factor-8). Readouts are analyzed by Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p≦0.05 as compared to the vehicle control group. [0321]
  • 3. Primary Antitumor Efficacy [0322]
  • 3.1. Early Therapy Models [0323]
  • 3.1.1. Subcutaneous Tumor [0324]
  • Tumor cells or frgments are implanted subcutaneously on [0325] Day 0. Vehicle and/or compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting at a time, usually on Day 1, prior to the ability to measure the tumor burden. Body weights and tumor measurements are recorded 2-3 times weekly. Mean net body and tumor weights are calculated for each data collection day. Anti-tumor efficacy may be initially determined by comparing the size of treated (T) and control (C) tumors on a given day by a Student's t-test, after the variance between groups is compared by an F-test, with significance determined at p≦0.05. The experiment may also be continued past the end of dosing in which case tumor measurements would continue to be recorded to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meirer curves from the times for individual tumors to attain the evaluation size. Significance is p≦0.05.
  • 3.1.2. Intraperitoneal/Intracranial Tumor Models [0326]
  • Tumor cells are injected intraperitoneally or intracranially on [0327] Day 0. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule starting on Day 1. Observations of morbidity and/or mortality are recorded twice daily. Body weights are measured and recorded twice weekly. Morbidity/mortality data is expressed in terms of the median time of survival and the number of long-term survivors is indicated separately. Survival times are used to generate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank test compared to the control group in the experiment.
  • 3.2. Established Disease Model [0328]
  • Tumor cells or fragments are implanted subcutaneously and grown to the desired size for treatment to begin. Once at the predetermined size range, mice are randomized into treatment groups. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value≦0.05 compared to the vehicle control group. [0329]
  • 3.3. Orthotopic Disease Models [0330]
  • 3.3.1. Mammary Fat Pad Assay [0331]
  • Tumor cells or fragments, of mammary adenocarcinoma origin, are implanted directly into a surgically exposed and reflected mammary fat pad in rodents. The fat pad is placed back in its original position and the surgical site is closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Tumor and body weights are measured and recorded 2-3 times weekly. Mean tumor weights of all groups over days post inoculation are graphed for comparison. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. [0332]
  • Tumor measurements may be recorded after dosing has stopped to monitor tumor growth delay. Tumor growth delays are expressed as the difference in the median time for the treated and control groups to attain a predetermined size divided by the median time for the control group to attain that size. Growth delays are compared by generating Kaplan-Meier curves from the times for individual tumors to attain the evaluation size. Significance is p value≦0.05 compared to the vehicle control group. In addition, this model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ, or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment. [0333]
  • 3.3.2. Intraprostatic Assay [0334]
  • Tumor cells or fragments, of prostatic adenocarcinoma origin, are implanted directly into a surgically exposed dorsal lobe of the prostate in rodents. The prostate is externalized through an abdominal incision so that the tumor can be implanted specifically in the dorsal lobe while verifying that the implant does not enter the seminal vesicles. The successfully inoculated prostate is replaced in the abdomen and the incisions through the abdomen and skin are closed. Hormones may also be administered to the rodents to support the growth of the tumors. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the lungs), or measuring the target organ weight (i.e., the regional lymph nodes). The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment. [0335]
  • 3.3.3. Intrabronchial Assay [0336]
  • Tumor cells of pulmonary origin may be implanted intrabronchially by making an incision through the skin and exposing the trachea The trachea is pierced with the beveled end of a 25 gauge needle and the tumor cells are inoculated into the main bronchus using a flat-ended 27 gauge needle with a 90° bend. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the contralateral lung), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment. [0337]
  • 3.3.4. Intracecal Assay [0338]
  • Tumor cells of gastrointestinal origin may be implanted intracecally by making an abdominal incision through the skin and externalizig the intestine. Tumor cells are inoculated into the cecal wall without penetrating the lumen of the intestine using a 27 or 30 gauge needle. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Body weights are measured and recorded 2-3 times weekly. At a predetermined time, the experiment is terminated and the animal is dissected. The size of the primary tumor is measured in three dimensions using either a caliper or an ocular micrometer attached to a dissecting scope. An F-test is preformed to determine if the variance is equal or unequal followed by a Student's t-test to compare tumor sizes in the treated and control groups at the end of treatment. Significance is p≦0.05 as compared to the control group. This model provides an opportunity to increase the rate of spontaneous metastasis of this type of tumor. Metastasis can be assessed at termination of the study by counting the number of visible foci per target organ (i.e., the liver), or measuring the target organ weight. The means of these endpoints are compared by Student's t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment. [0339]
  • 4. Secondary (Metastatic) Antitumor Efficacy [0340]
  • 4.1. Spontaneous Metastasis [0341]
  • Tumor cells are inoculated s.c. and the tumors allowed to grow to a predetermined range for spontaneous metastasis studies to the lung or liver. These primary tumors are then excised. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule which may include the period leading up to the excision of the primary tumor to evaluate therapies directed at inhibiting the early stages of tumor metastasis. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplah-Meier curves. Significance is p≦0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance determined at p≦0.05 compared to the control group in the experiment for both of these endpoints. [0342]
  • 4.2. Forced Metastasis [0343]
  • Tumor cells are injected into the tail vein, portal vein, or the left ventricle of the heart in experimental (forced) lung, liver, and bone metastasis studies, respectively. Compounds are administered p.o., i.p., i.v., i.m., or s.c. according to a predetermined schedule. Observations of morbidity and/or mortality are recorded daily. Body weights are measured and recorded twice weekly. Potential endpoints include survival time, numbers of visible foci per target organ, or target organ weight. When survival time is used as the endpoint the other values are not determined. Survival data is used to generate Kaplan-Meier curves. Significance is p≦0.05 by a log-rank test compared to the control group in the experiment. The mean number of visible tumor foci, as determined under a dissecting microscope, and the mean target organ weights are compared by Student's t-test after conducting an F-test, with significance at p≦0.05 compared to the vehicle control group in the experiment for both endpoints. [0344]
    • EXAMPLE 7
  • Proliferation Inhibition Assay: Antisense Oligonucleotides Suppress the Growth of Cancer Cell Lines [0345]
  • The cell line used for testing is the human colon cancer cell line HCT116. Cells are cultured in RPMI-1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml and kept at 37° C. in a 95% air/5%CO[0346] 2 atmosphere.
  • Phosphorothioate oligoribonucleotides are synthesized on an Applied Biosystems Model 380B DNA synthesizer using phosphoroamnidite chemistry. A sequence of 24 bases complementary to the nucleotides at [0347] position 1 to 24 of SEQ ID NO:1 is used as the test oligonucleotide. As a control, another (random) sequence is used: 5′-TCA ACT GAC TAG ATG TAC ATG GAC-3′. Following assembly and deprotection, oligonucleotides are ethanol-precipitated twice, dried, and suspended in phosphate buffered saline at the desired concentration. Purity of the oligonucleotides is tested by capillary gel electrophoresis and ion exchange HPLC. The purified oligonucleotides are added to the culture medium at a concentration of 10 μM once per day for seven days.
  • The addition of the test oligonucleotide for seven days results in significantly reduced expression of human transient receptor potential channel as determined by Western blotting. This effect is not observed with the control oligonucleotide. After 3 to 7 days, the number of cells in the cultures is counted using an automatic cell counter. The number of cells in cultures treated with the test oligonucleotide (expressed as 100%) is compared with the number of cells in cultures treated with the control oligonucleotide. The number of cells in cultures treated with the test oligonucleotide is not more than 30% of control, indicating that the inhibition of human transient receptor potential channel has an anti-proliferative effect on cancer cells. [0348]
    • EXAMPLE 8
  • In vivo Testing of Compounds/target Validation [0349]
  • 1. Pain: [0350]
  • Acute Pain [0351]
  • Acute pain is measured on a hot plate mainly in rats. Two variants of hot plate testing are used: In the classical variant animals are put on a hot surface (52 to 56° C.) and the latency time is measured until the animals show nocifensive behavior, such as stepping or foot licking. The other variant is an increasing temperature hot plate where the experimental animals are put on a surface of neutral temperature. Subsequently this surface is slowly but constantly heated until the animals begin to lick a hind paw. The temperature which is reached when hind paw licking begins is a measure for pain threshold. [0352]
  • Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing. [0353]
  • Persistent Pain [0354]
  • Persistent pain is measured with the formalin or capsaicin test, mainly in rats. A solution of 1 to 5% formalin or 10 to 100 μg capsaicin is injected into one hind paw of the experimental animal. After formalin or capsaicin application the animals show nocifensive reactions like flinching, licking and biting of the affected paw. The number of nocifensive reactions within a time frame of up to 90 minutes is a measure for intensity of pain. [0355]
  • Compounds are tested against a vehicle treated control group. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradernal, transdermal) prior to formalin or capsaicin administration. [0356]
  • Neuropathic Pain [0357]
  • Neuropathic pain is induced by different variants of unilateral sciatic nerve injury mainly in rats. The operation is performed under anesthesia. The first variant of sciatic nerve injury is produced by placing loosely constrictive ligatures around the common sciatic nerve. The second variant is the tight ligation of about the half of the diameter of the common sciatic nerve. In the next variant, a group of models is used in which tight ligations or transections are made of either the L5 and L6 spinal nerves, or the L% spinal nerve only. The fourth variant involves an axotomy of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact whereas the last variant comprises the axotomy of only the tibial branch leaving the sural and common nerves uninjured. Control animals are treated with a sham operation. [0358]
  • Postoperatively, the nerve injured animals develop a chronic mechanical allodynia, cold allodynioa, as well as a thermal hyperalgesia Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc.-Life Science Instruments, Woodland Hills, SA, USA; Electronic von Frey System, Somedic Sales AB, Hörby, Sweden). Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy), or by means of a cold plate of 5 to 10° C. where the nocifensive reactions of the affected hind paw are counted as a measure of pain intensity. A further test for cold induced pain is the counting of nocifensive reactions, or duration of nocifensive responses after plantar administration of acetone to the affected hind limb. Chronic pain in general is assessed by registering the circadanian rhythms in activity (Surjo and Arndt, Universität zu Köln, Cologne, Germany), and by scoring differences in gait (foot print patterns; FOOTPRINTS program, Klapdor et al., 1997. A low cost method to analyze footprint patterns. J. Neurosci. Methods 75, 49-54). [0359]
  • Compounds are tested against sham operated and vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing. [0360]
  • Inflammatory Pain [0361]
  • Inflammatory pain is induced mainly in rats by injection of 0.75 mg carrageenan or complete Freund's adjuvant into one hind paw. The animals develop an edema with mechanical allodynia as well as thermal hyperalgesia. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc.-Life Science Instruments, Woodland Hills, SA, USA). Thermal hyperalgesia is measured by means of a radiant heat source (Plantar Test, Ugo Basile, Comerio, Italy, Paw thermal stimulator, G. Ozaki, University of California, USA). For edema measurement two methods are being used. In the first method, the animals are sacrificed and the affected hindpaws sectioned and weighed. The second method comprises differences in paw volume by measuring water displacement in a plethysmometer (Ugo Basile, Comerio, Italy). [0362]
  • Compounds are tested against uninflamed as well as vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing. [0363]
  • Diabetic Neuropathic Pain [0364]
  • Rats treated with a single intraperitoneal injection of 50 to 80 mg/kg streptozotocin develop a profound hyperglycemia and mechanical allodynia within 1 to 3 weeks. Mechanical allodynia is measured by means of a pressure transducer (electronic von Frey Anesthesiometer, IITC Inc.-Life Science Instruments, Woodland Hills, SA, USA). [0365]
  • Compounds are tested against diabetic and non-diabetic vehicle treated control groups. Substance application is performed at different time points via different application routes (i.v., i.p., p.o., i.t., i.c.v., s.c., intradermal, transdermal) prior to pain testing. [0366]
  • [0367] 2. Parkinson's Disease
  • 6-Hydroxydopamine (6-OH-DA) Lesion [0368]
  • Degeneration of the dopaminergic nigrostriatal and striatopallidal pathways is the central pathological event in Parkinson's disease. This disorder has been mimicked experimnentally in rats using single/sequential unilateral stereotaxic injections of 6-OH-DA into the medium forebrain bundle (MFB). [0369]
  • Male Wistar rats (Harlan Winkelmann, Germany), weighing 200±250 g at the beginning of the experiment, are used. The rats are maintained in a temperature- and humidity-controlled environment under a 12 h light/dark cycle with free access to food and water when not in experimental sessions. The following in vivo protocols are approved by the governmental authorities. All efforts are made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques. [0370]
  • Animals are administered pargyline on the day of surgery (Sigma, St. Louis, Mo., USA; 50 mg/kg i.p.) in order to inhibit metabolism of 6-OHDA by monoamine oxidase and desmethyliiipramine HCl (Sigma; 25 mg/kg i.p.) in order to prevent uptake of 6-OHDA by noradrenergic terminals. Thirty minutes later the rats are anesthetized with sodium pentobarbital (50 mg/kg) and placed in a stereotaxic frame. In order to lesion the DA [0371] nigrostriatal pathway 4 μl of 0.01% ascorbic acid-saline containing 8 μg of 6-OHDA HBr (Sigma) are injected into the left medial fore-brain bundle at a rate of 1 μl/min (2.4 mm anterior, 1.49 mm lateral, −2.7 mm ventral to Bregma and the skull surface). The needle is left in place an additional 5 min to allow diffusion to occur.
  • Stepping Test [0372]
  • Forelimb akinesia is assessed three weeks following lesion placement using a modified stepping test protocol. In brief, the animals are held by the experimenter with one hand fixing the hindlimbs and slightly raising the hind part above the surface. One paw is touching the table, and is then moved slowly sideways (5 s for 1 m), first in the forehand and then in the backhand direction. The number of adjusting steps is counted for both paws in the backhand and forehand direction of movement. The sequence of testing is right paw forehand and backhand adjusting stepping, followed by left paw forehand and backhand directions. The test is repeated three times on three consecutive days, after an initial training period of three days prior to the first testing. Forehand adjusted stepping reveals no consistent differences between lesioned and healthy control animals. Analysis is therefore restricted to backhand adjusted stepping. [0373]
  • Balance Test [0374]
  • Balance adjustments following postural challenge are also measured during the stepping test sessions. The rats are held in the same position as described in the stepping test and, instead of being moved sideways, tilted by the experimenter towards the side of the paw touching the table. This maneuver results in loss of balance and the ability of the rats to regain balance by forelimb movements is scored on a scale ranging from 0 to 3. [0375] Score 0 is given for a normal forelimb placement. When the forelimb movement is delayed but recovery of postural balance detected, score 1 is given. Score 2 represents a clear, yet insufficient, forelimb reaction, as evidenced by muscle contraction, but lack of success in recovering balance, and score 3 is given for no reaction of movement. The test is repeated three times a day on each side for three consecutive days after an initial training period of three days prior to the first testing.
  • Staircase Test (Paw Reaching) [0376]
  • A modified version of the staircase test is used for evaluation of paw reaching behavior three weeks following primary and secondary lesion placement. Plexiglass test boxes with a central platform and a removable staircase on each side are used. The apparatus is designed such that only the paw on the same side at each staircase can be used, thus providing a measure of independent forelimb use. For each test the animals are left in the test boxes for 15 min. The double staircase is filled with 7×3 chow pellets (Precision food pellets, formula: P, purified rodent diet, size 45 mg; Sandown Scientific) on each side. After each test the number of pellets eaten (successfully retrieved pellets) and the number of pellets taken (touched but dropped) for each paw and the success rate (pellets eaten/pellets taken) are counted separately. After three days of food deprivation (12 g per animal per day) the animals are tested for 11 days. Full analysis is conducted only for the last five days. [0377]
  • MPTP Treatment [0378]
  • The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) causes degeneration of mesencephalic dopaminergic (DAergic) neurons in rodents, non-human primates, and humans and, in so doing, reproduces many of the symptoms of Parkinson's disease. MPTP leads to a marked decrease in the levels of dopamine and its metabolites, and in the number of dopaminergic terminals in the striatum as well as severe loss of the tyrosine hydroxylase (TH)-immunoreactive cell bodies in the substantia nigra, pars compacta. [0379]
  • In order to obtain severe and long-lasting lesions, and to reduce mortality, animals receive single injections of MPTP, and are then tested for severity of lesion 7-10 days later. Successive MPTP injections are administered on [0380] days 1, 2 and 3. Animals receive application of 4 mg/kg MPNT hydrochloride (Sigma) in saline once daily. All injections are intraperitoneal (i.p.) and the MPTP stock solution is frozen between injections. Animals are decapitated on day 11.
  • Immunohistology [0381]
  • At the completion of behavioral experiments, all animals are anaesthetized with 3 ml thiopental (1 g/40 ml i.p., Tyrol Pharma). The mice are perfused transcardially with 0.01 M PBS (pH 7.4) for 2 min, followed by 4% paraformaldehyde (Merck) in PBS for 15 min. The brains are removed and placed in 4% paraformaldehyde for 24 h at 4° C. For dehydration they are then transferred to a 20% sucrose (Merck) solution in 0.1 M PBS at 4° C. until they sink. The brains are frozen in methylbutan at −20° C. for 2 min and stored at −70° C. Using a sledge microtome (mod. 3800-Frigocut, Leica), 25 μm sections are taken from the genu of the corpus callosum (AP 1.7 mm) to the hippocampus (AP 21.8 mm) and from AP 24.16 to AP 26.72. Forty-six sections are cut and stored in assorters in 0.25 M Tris buffer (pH 7.4) for immunohistochernistry. [0382]
  • A series of sections is processed for free-floating tyrosine hydroxylase (TH) immunohistochemistry. Following three rinses in 0.1 M PBS, endogenous peroxidase activity is quenched for 10 min in 0.3% H[0383] 2O2 ±PBS. After rinsing in PBS, sections are preincubated in 10% normal bovine serum (Sigma) for 5 min as blocking agent and transferred to either primary anti-rat TH rabbit antiserum (dilution 1:2000).
  • Following overnight incubation at room temperature, sections for TH immunoreactivity are rinsed in PBS (2×10 min) and incubated in biotinylated anti-rabbit immunoglobulin G raised in goat (dilution 1:200) (Vector) for 90 min, rinsed repeatedly and transferred to Vectastain ABC (Vector) solution for 1 h. 3,.3′-Diaminobenzidine tetrahydrochloride (DAB; Sigma) in 0.1 M PBS, supplemented with 0.005% H[0384] 2O2, serves as chromogen in the subsequent visualization reaction. Sections are mounted on to gelatin-coated slides, left to dry overnight, counter-stained with hematoxylin dehydrated in ascending alcohol concentrations and cleared in butylacetate. Coverslips are mounted on entellan.
  • Rotarod Test [0385]
  • We use a modification of the procedure described by Rozas and Labandeira-Garcia (1997), with a CR-1 Rotamex system (Columbus Instruments, Columbus, Ohio) comprising an IBM-compatible personal computer, a CIO-24 data acquisition card, a control unit, and a four-lane rotarod unit. The rotarod unit consists of a rotating spindle (diameter 7.3 cm) and individual compartments for each mouse. The system software allows preprogramming of session protocols with varying rotational speeds (0-80 rpm). Infrared beams are used to detect when a mouse has fallen onto the base grid beneath the rotarod. The system logs the fall as the end of the experiment for that mouse, and the total time on the rotarod, as well as the time of the fall and all the set-up parameters, are recorded. The system also allows a weak current to be passed through the base grid, to aid training. [0386]
  • 3. Dementia [0387]
  • The Object Recognition Task [0388]
  • The object recognition task has been designed to assess the effects of experimental manipulations on the cognitive performance of rodents. A rat is placed in an open field, in which two identical objects are present. The rats inspects both objects during the first trial of the object recognition task. In a second trial, after a retention interval of for example 24 hours, one of the two objects used in the first trial, the ‘familiar’ object, and a novel object are placed in the open field. The inspection time at each of the objects is registered. The basic measures in the OR task is the time spent by a rat exploring the two object the second trial. Good retention is reflected by higher exploration times towards the novel than the ‘familiar’ object. [0389]
  • Administration of the putative cognition enhancer prior to the first trial predominantly allows assessment of the effects on acquisition, and eventually on consolidation processes. Administration of the testing compound after the first trial allows to assess the effects on consolidation processes, whereas administration before the second trial allows to measure effects on retrieval processes. [0390]
  • The Passive Avoidance Task [0391]
  • The passive avoidance task assesses memory performance in rats and mice. The inhibitory avoidance apparatus consists of a two-compartment box with a light compartment and a dark compartment. The two compartments are separated by a guillotine door that can be operated by the experimenter. A threshold of 2 cm separates the two compartments when the guillotine door is raised. When the door is open, the illumination in the dark compartment is about 2 lux. The light intensity is about 500 lux at the center of the floor of the light compartment. [0392]
  • Two habituation sessions, one shock session, and a retention session are given, separated by inter-session intervals of 24 hours. In the habituation sessions and the retention session the rat is allowed to explore the apparatus for 300 sec. The rat is placed in the light compartment, facing the wall opposite to the guillotine door. After an accommodation period of 15 sec. the guillotine door is opened so that all parts of the apparatus can be visited freely. Rats normally avoid brightly lit areas and will enter the dark compartment within a few seconds. [0393]
  • In the shock session the guillotine door between the compartments is lowered as soon as the rat has entered the dark compartment with its four paws, and a scrambled 1 mA footshock is administered for 2 sec. The rat is removed from the apparatus and put back into its home cage. The procedure during the retention session is identical to that of the habituation sessions. [0394]
  • The step-through latency, that is the first latency of entering the dark compartment (in sec.) during the retention session is an index of the memory performance of the animal; the longer the latency to enter the dark compartment, the better the retention is. A testing compound in given half an hour before the shock session, together with 1 mg*kg[0395] −1 scopolamine. Scopolamine impairs the memory performance during the retention session 24 hours later. If the test compound increases the enter latency compared with the scopolamine-treated controls, is likely to possess cognition enhancing potential.
  • The Morris Water Escape Task [0396]
  • The Morris water escape task measures spatial orientation learning in rodents. It is a test system that has extensively been used to investigate the effects of putative therapeutic on the cognitive functions of rats and mice. The performance of an animal is assessed in a circular water tank with an escape platform that is submerged about 1 cm below the surface of the water. The escape platform is not visible for an animal swimming in the water tank. Abundant extra-maze cues are provided by the furniture in the room, including desks, computer equipment, a second water tank, the presence of the experimenter, and by a radio on a shelf that is playing softly. [0397]
  • The animals receive four trials during five daily acquisition sessions. A trial is started by placing an animal into the pool, facing the wall of the tank. Each of four starting positions in the quadrants north, east, south, and west is used once in a series of four trials; their order is randomized. The escape platform is always in the same position. A trial is terminated as soon as the animal had climbs onto the escape platform or when 90 seconds have elapsed, whichever event occurs first. The animal is allowed to stay on the platform for 30 seconds. Then it is taken from the platform and the next trial is started. If an animal did not find the platform within 90 seconds it is put on the platform by the experimenter and is allowed to stay there for 30 seconds. After the fourth trial of the fifth daily session, an additional trial is given as a probe trial: the platform is removed, and the time the animal spends in the four quadrants is measured for 30 or 60 seconds. In the probe trial, all animals start from the same start position, opposite to the quadrant where the escape platform had been positioned during acquisition. [0398]
  • Four different measures are taken to evaluate the performance of an animal during acquisition training: escape latency, traveled distance, distance to platform, and swimming speed. The following measures are evaluated for the probe trial: time (s) in quadrants and traveled distance (cm) in the four quadrants. The probe trial provides additional information about how well an animal learned the position of the escape platform. If an animal spends more time and swims a longer distance in the quadrant where the platform had been positioned during the acquisition sessions than in any other quadrant, one concludes that the platform position has been learned well. [0399]
  • In order to assess the effects of putative cognition enhancing compounds, rats or mice with specific brain lesions which impair cognitive functions, or animals treated with compounds such as scopolamine or MK-801, which interfere with normal learning, or aged animals which suffer from cognitive deficits, are used. [0400]
  • The T-maze Spontaneous Alternation Task [0401]
  • The T-maze spontaneous alternation task (TeMCAT) assesses the spatial memory performance in mice. The start arm and the two goal arms of the T-maze are provided with guillotine doors which can be operated manually by the experimenter. A mouse is put into the start arm at the beginning of training. The guillotine door is closed. In the first trial, the ‘forced trial’, either the left or right goal arm is blocked by lowering the guillotine door. After the mouse has been released from the start arm, it will negotiate the maze, eventually enter the open goal arm, and return to the start position, where it will be confined for 5 seconds, by lowering the guillotine door. Then, the animal can choose freely between the left and right goal arm (all guillotine-doors opened) during 14 ‘free choice’ trials. As soon a the mouse has entered one goal arm, the other one is closed. The mouse eventually returns to the start arm and is free to visit whichever go alarm it wants after having been confined to the start arm for 5 seconds. After completion of 14 free choice trials in one session, the animal is removed from the maze. During training, the animal is never handled. [0402]
  • The percent alternations out of 14 trials is calculated. This percentage and the total time needed to complete the first forced trial and the subsequent 14 free choice trials (in s) is analyzed. Cognitive deficits are usually induced by an injection of scopolamine, 30 min before the start of the training session. Scopolamine reduced the per-cent alternations to chance level, or below. A cognition enhancer, which is always administered before the training session, will at least partially, antagonize the scopolamine-induced reduction in the spontaneous alternation rate. [0403]
  • References [0404]
  • 1. From worm to man: three subfamilies of TRP channels. Harteneck C, Plant TD, Schultz G, Trends Neurosci 2000 Apr;23(4):159-66 [0405]
  • 2. Cloning, expression and subcellular localization of two novel splice variants of mouse transient receptor [0406] potential channel 2, Hofmann T, Schaefer M, Schultz G, Gudermann T, Biochem J 2000 Oct 1;351(Pt 1):115-122
  • 3. Cloning and expression of the human transient receptor potential 4 (TRP4) gene: [0407]
  • localization and functional expression of human TRP4 and TRP3. McKay RR, Szymeczek-Seay CL, Lievremont JP, Bird [0408]
  • GS, Zitt C, Jungling E, Luckhoff A, Putney Jr JW, Biochem J 2000 [0409] Nov 1;351 Pt 3:735-46
  • 4. Cloning and expression of the human transient receptor potential 4 (TRP4) gene: [0410]
  • Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel. Sun M, [0411]
  • Goldin E, Stahl S, Falardeau JL, Kennedy JC, Aciemo JS Jr, Bove C, Kaneski CR, Nagle J, Bromley MC, Cohnan M, Schifftnann R, Slaugenhaupt SA, Hum Mol Genet 2000 [0412] Oct 12;9(17):2471-8
  • 5. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T, Schultz G, Nature 1999 [0413] Jan 21;397(6716):259-63
  • 6. Identification and characterization of MIR1, a novel gene with homology to melastatin (MLSN1) and the trp gene family located in the BWS-WT2 critical region on chromosome 11p15.5 and showing allele-specific expression. Prawitt D, Enklaar T, Klemm G, Gartner B, Spangenberg C, Winterpacht A, Higgins M, Pelletier J, Zabel B, Hum Mol Genet 2000 [0414] Jan 22;9(2):203-16
  • 7. Evidence for a role of Trp proteins in the oxidative stress-induced membrane conductances of porcine aortic endothelial cells. Balzer M, Lintschinger B, Groschner K, Cardiovasc Res 1999 [0415]
  • May;42(2):543-9 8. Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5. Schaefer M, Plant TD, Obukhov AG, Hofiann T, Gudermann T, Schultz G,: J Biol Chem 2000 [0416] Jun 9;275(23):17517-26
  • 1 12 1 3402 DNA Homo sapiens 1 atgctgcgac ctcaggtgaa agaggagatc atctgcatga ttcagaacac tttcaacttt 60 agtcttaaac agtccaagca ccttttccaa attctaatgg agtgtatggt tcacagggat 120 tgtattacca tatttgatgc tgactctgaa gagcagcaag acctggactt agcaatccta 180 acagctttgc tgaagggcac aaatttatca gcgtcagagc aattaaatct ggcaatggct 240 tgggacaggg tggacattgc caagaaacat atcctaattt atgaacaaca ctggaagcct 300 gatgccctgg aacaagcaat gtcagatgct ttagtgatgg atcgggtgga ttttgtgaag 360 ctcttaatag aatatggagt gaacctccat cgctttctta ccatccctcg actggaagag 420 ctctacaata caaaacaagg acctactaat acactcttgc atcatctcgt ccaagatgtg 480 aaacagcata cccttctttc aggctaccga ataaccttga ttgacattgg attagtagta 540 gaatacctca ttggtagagc atatcgcagc aactacacta gaaaacattt cagagccctc 600 tacaacaacc tctacagaaa atacaagcac cagagacact cctcaggaaa tagaaatgag 660 tctgcagaaa gtacgctgca ctcccagttc attagaactg cacagccata caaattcaag 720 gaaaagtcta tagtccttca taaatcaagg aagaagtcaa aagaacaaaa tgtatcagat 780 gaccctgagt ctactggctt tctttaccct tacaatgacc tgctggtttg ggctgtgctg 840 atgaaaaggc agaagatggc tatgttcttc tggcagcatg gagaggaggc cacggttaaa 900 gccgtgattg cgtgtatcct ctaccgggca atggcccatg aagctaagga gagtcacatg 960 gtggatgatg cctcagaaga gttgaagaat tactcaaaac agtttggcca gctggctctg 1020 gacttgttgg agaaggcatt caagcagaat gagcgcatgg ccatgacgct gttgacgtat 1080 gaactcagga actggagcaa ttcgacctgc cttaaactgg ccgtgtcggg aggattacga 1140 ccctttgttt cacatacttg tacccagatg ctactgacag acatgtggat ggggaggctg 1200 aaaatgagga aaaactcttg gttaaagatt attataagca ttattttacc acccaccatt 1260 ttgacactgg aatttaaaag caaagctgag atgtcacatg ttccccagtc ccaggacttc 1320 caatttatgt ggtattacag tgaccagaac gccagcagtt ccaaagaaag tgcttctgtg 1380 aaagagtatg atttggaaag gggccatgat gagaaactgg atgaaaatca gcattttggt 1440 ttggaaagtg ggcaccaaca ccttccgtgg accaggaaag tctatgagtt ctacagtgct 1500 ccaattgtca agttttggtt ttatacgatg gcgtatttgg cattcctcat gctgttcact 1560 tacaccgtgt tggtggagat gcagccccag cccagcgtgc aggagtggct tgttagcatt 1620 tacatcttca ccaatgctat tgaggtggtc agggagatct gtatttcaga acctgggaag 1680 tttacccaaa aggtgaaggt atggattagt gagtactgga acttaacaga aactgtggcc 1740 attggcctgt tttcagctgg cttcgtcctt cgatggggtg accctccttt tcacacagcg 1800 ggaagactga tctactgcat agacatcata ttctggttct cacggctcct ggacttcttt 1860 gctgtgaatc aacatgcagg tccatatgtg accatgattg caaaaatgac agcaaacatg 1920 ttctatattg tgatcatcat ggccatagtc ctgctgagct ttggagtggc acgcaaggcc 1980 atcctttcgc caaaagagcc accatcttgg agtctagctc gagatattgt atttgagcca 2040 tactggatga tatacggaga agtctatgct ggagaaatag atgtttgttc aagccagcca 2100 tcctgccctc ctggttcttt tcttactcca ttcttgcaag ctgtctacct cttcgtgcaa 2160 tatatcatca tggtgaacct gttgattgct ttcttcaaca acgtttactt agatatggaa 2220 tccatttcaa ataacctgtg gaaatacaac cgctatcgct acatcatgac ctaccacgag 2280 aagccctggc tgcccccacc tctcatcctg ctgagccacg tgggccttct cctccgccgc 2340 ctgtgctgtc atcgagctcc tcacgaccag gaagagggtg acgttggatt aaaactctac 2400 ctcagtaagg aggatctgaa aaaacttcat gattttgagg agcagtgcgt ggaaaaatac 2460 ttccatgaga agatggaaga tgtgaattgt agttgtgagg aacgaatccg agtgacatca 2520 gaaagggtta cagagatgta cttccagctg aaagaaatga atgaaaaggt gtcttttata 2580 aaggactcct tactgtcttt ggacagccag gtgggacacc tgcaggatct ctctgccctg 2640 actgtggata ccctgaaagt cctttctgct gttgacactt tgcaagagga tgaggctctc 2700 ctggccaaga gaaagcattc tacttgcaaa aaacttcccc acagctggag caatgtcatc 2760 tgtgcagagg ttctaggcag catggagatc gctggagaga agaaatacca gtattatagc 2820 atgccctctt ctttgctgag gagcctggct ggaggccggc atcccccaag agtgcagagg 2880 ggggcacttc ttgagattac aaacagtaaa agagaggcta caaatgtaag aaatgaccag 2940 gaaaggcaag aaacacaaag tagtatagtg gtttctgggg tgtctcctaa caggcaagca 3000 cactcaaagt atggccagtt tcttctggtc ccctctaatc taaagcgagt tcctttttca 3060 gcagaaactg tcttgcctct gtccagaccc tctgtgccag atgtgctggc aactgaacag 3120 gacatccaga ctgaggttct tgttcatctg actgggcaga ccccaattgt ctctgactgg 3180 gcatcagtgg atgaacccaa ggaaaagcac gagcctattg ctcacttact ggatggacaa 3240 gacaaggcag agcaagtgct acccactttg agttgcacac ctgaacccat gacaatgagc 3300 tcccctctcc gcagatacag gcccttcgct aggagtcata gttttagatt ccataaggag 3360 gagaaattga tgaagatctg taagattaaa agtaaggaat aa 3402 2 1133 PRT Homo sapiens 2 Met Leu Arg Pro Gln Val Lys Glu Glu Ile Ile Cys Met Ile Gln Asn 1 5 10 15 Thr Phe Asn Phe Ser Leu Lys Gln Ser Lys His Leu Phe Gln Ile Leu 20 25 30 Met Glu Cys Met Val His Arg Asp Cys Ile Thr Ile Phe Asp Ala Asp 35 40 45 Ser Glu Glu Gln Gln Asp Leu Asp Leu Ala Ile Leu Thr Ala Leu Leu 50 55 60 Lys Gly Thr Asn Leu Ser Ala Ser Glu Gln Leu Asn Leu Ala Met Ala 65 70 75 80 Trp Asp Arg Val Asp Ile Ala Lys Lys His Ile Leu Ile Tyr Glu Gln 85 90 95 His Trp Lys Pro Asp Ala Leu Glu Gln Ala Met Ser Asp Ala Leu Val 100 105 110 Met Asp Arg Val Asp Phe Val Lys Leu Leu Ile Glu Tyr Gly Val Asn 115 120 125 Leu His Arg Phe Leu Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn Thr 130 135 140 Lys Gln Gly Pro Thr Asn Thr Leu Leu His His Leu Val Gln Asp Val 145 150 155 160 Lys Gln His Thr Leu Leu Ser Gly Tyr Arg Ile Thr Leu Ile Asp Ile 165 170 175 Gly Leu Val Val Glu Tyr Leu Ile Gly Arg Ala Tyr Arg Ser Asn Tyr 180 185 190 Thr Arg Lys His Phe Arg Ala Leu Tyr Asn Asn Leu Tyr Arg Lys Tyr 195 200 205 Lys His Gln Arg His Ser Ser Gly Asn Arg Asn Glu Ser Ala Glu Ser 210 215 220 Thr Leu His Ser Gln Phe Ile Arg Thr Ala Gln Pro Tyr Lys Phe Lys 225 230 235 240 Glu Lys Ser Ile Val Leu His Lys Ser Arg Lys Lys Ser Lys Glu Gln 245 250 255 Asn Val Ser Asp Asp Pro Glu Ser Thr Gly Phe Leu Tyr Pro Tyr Asn 260 265 270 Asp Leu Leu Val Trp Ala Val Leu Met Lys Arg Gln Lys Met Ala Met 275 280 285 Phe Phe Trp Gln His Gly Glu Glu Ala Thr Val Lys Ala Val Ile Ala 290 295 300 Cys Ile Leu Tyr Arg Ala Met Ala His Glu Ala Lys Glu Ser His Met 305 310 315 320 Val Asp Asp Ala Ser Glu Glu Leu Lys Asn Tyr Ser Lys Gln Phe Gly 325 330 335 Gln Leu Ala Leu Asp Leu Leu Glu Lys Ala Phe Lys Gln Asn Glu Arg 340 345 350 Met Ala Met Thr Leu Leu Thr Tyr Glu Leu Arg Asn Trp Ser Asn Ser 355 360 365 Thr Cys Leu Lys Leu Ala Val Ser Gly Gly Leu Arg Pro Phe Val Ser 370 375 380 His Thr Cys Thr Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu 385 390 395 400 Lys Met Arg Lys Asn Ser Trp Leu Lys Ile Ile Ile Ser Ile Ile Leu 405 410 415 Pro Pro Thr Ile Leu Thr Leu Glu Phe Lys Ser Lys Ala Glu Met Ser 420 425 430 His Val Pro Gln Ser Gln Asp Phe Gln Phe Met Trp Tyr Tyr Ser Asp 435 440 445 Gln Asn Ala Ser Ser Ser Lys Glu Ser Ala Ser Val Lys Glu Tyr Asp 450 455 460 Leu Glu Arg Gly His Asp Glu Lys Leu Asp Glu Asn Gln His Phe Gly 465 470 475 480 Leu Glu Ser Gly His Gln His Leu Pro Trp Thr Arg Lys Val Tyr Glu 485 490 495 Phe Tyr Ser Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr Met Ala Tyr 500 505 510 Leu Ala Phe Leu Met Leu Phe Thr Tyr Thr Val Leu Val Glu Met Gln 515 520 525 Pro Gln Pro Ser Val Gln Glu Trp Leu Val Ser Ile Tyr Ile Phe Thr 530 535 540 Asn Ala Ile Glu Val Val Arg Glu Ile Cys Ile Ser Glu Pro Gly Lys 545 550 555 560 Phe Thr Gln Lys Val Lys Val Trp Ile Ser Glu Tyr Trp Asn Leu Thr 565 570 575 Glu Thr Val Ala Ile Gly Leu Phe Ser Ala Gly Phe Val Leu Arg Trp 580 585 590 Gly Asp Pro Pro Phe His Thr Ala Gly Arg Leu Ile Tyr Cys Ile Asp 595 600 605 Ile Ile Phe Trp Phe Ser Arg Leu Leu Asp Phe Phe Ala Val Asn Gln 610 615 620 His Ala Gly Pro Tyr Val Thr Met Ile Ala Lys Met Thr Ala Asn Met 625 630 635 640 Phe Tyr Ile Val Ile Ile Met Ala Ile Val Leu Leu Ser Phe Gly Val 645 650 655 Ala Arg Lys Ala Ile Leu Ser Pro Lys Glu Pro Pro Ser Trp Ser Leu 660 665 670 Ala Arg Asp Ile Val Phe Glu Pro Tyr Trp Met Ile Tyr Gly Glu Val 675 680 685 Tyr Ala Gly Glu Ile Asp Val Cys Ser Ser Gln Pro Ser Cys Pro Pro 690 695 700 Gly Ser Phe Leu Thr Pro Phe Leu Gln Ala Val Tyr Leu Phe Val Gln 705 710 715 720 Tyr Ile Ile Met Val Asn Leu Leu Ile Ala Phe Phe Asn Asn Val Tyr 725 730 735 Leu Asp Met Glu Ser Ile Ser Asn Asn Leu Trp Lys Tyr Asn Arg Tyr 740 745 750 Arg Tyr Ile Met Thr Tyr His Glu Lys Pro Trp Leu Pro Pro Pro Leu 755 760 765 Ile Leu Leu Ser His Val Gly Leu Leu Leu Arg Arg Leu Cys Cys His 770 775 780 Arg Ala Pro His Asp Gln Glu Glu Gly Asp Val Gly Leu Lys Leu Tyr 785 790 795 800 Leu Ser Lys Glu Asp Leu Lys Lys Leu His Asp Phe Glu Glu Gln Cys 805 810 815 Val Glu Lys Tyr Phe His Glu Lys Met Glu Asp Val Asn Cys Ser Cys 820 825 830 Glu Glu Arg Ile Arg Val Thr Ser Glu Arg Val Thr Glu Met Tyr Phe 835 840 845 Gln Leu Lys Glu Met Asn Glu Lys Val Ser Phe Ile Lys Asp Ser Leu 850 855 860 Leu Ser Leu Asp Ser Gln Val Gly His Leu Gln Asp Leu Ser Ala Leu 865 870 875 880 Thr Val Asp Thr Leu Lys Val Leu Ser Ala Val Asp Thr Leu Gln Glu 885 890 895 Asp Glu Ala Leu Leu Ala Lys Arg Lys His Ser Thr Cys Lys Lys Leu 900 905 910 Pro His Ser Trp Ser Asn Val Ile Cys Ala Glu Val Leu Gly Ser Met 915 920 925 Glu Ile Ala Gly Glu Lys Lys Tyr Gln Tyr Tyr Ser Met Pro Ser Ser 930 935 940 Leu Leu Arg Ser Leu Ala Gly Gly Arg His Pro Pro Arg Val Gln Arg 945 950 955 960 Gly Ala Leu Leu Glu Ile Thr Asn Ser Lys Arg Glu Ala Thr Asn Val 965 970 975 Arg Asn Asp Gln Glu Arg Gln Glu Thr Gln Ser Ser Ile Val Val Ser 980 985 990 Gly Val Ser Pro Asn Arg Gln Ala His Ser Lys Tyr Gly Gln Phe Leu 995 1000 1005 Leu Val Pro Ser Asn Leu Lys Arg Val Pro Phe Ser Ala Glu Thr 1010 1015 1020 Val Leu Pro Leu Ser Arg Pro Ser Val Pro Asp Val Leu Ala Thr 1025 1030 1035 Glu Gln Asp Ile Gln Thr Glu Val Leu Val His Leu Thr Gly Gln 1040 1045 1050 Thr Pro Ile Val Ser Asp Trp Ala Ser Val Asp Glu Pro Lys Glu 1055 1060 1065 Lys His Glu Pro Ile Ala His Leu Leu Asp Gly Gln Asp Lys Ala 1070 1075 1080 Glu Gln Val Leu Pro Thr Leu Ser Cys Thr Pro Glu Pro Met Thr 1085 1090 1095 Met Ser Ser Pro Leu Arg Arg Tyr Arg Pro Phe Ala Arg Ser His 1100 1105 1110 Ser Phe Arg Phe His Lys Glu Glu Lys Leu Met Lys Ile Cys Lys 1115 1120 1125 Ile Lys Ser Lys Glu 1130 3 1503 PRT Homo sapiens 3 Met Glu Pro Ser Ala Leu Arg Lys Ala Gly Ser Glu Gln Glu Glu Gly 1 5 10 15 Phe Glu Gly Leu Pro Arg Arg Val Thr Asp Leu Gly Met Val Ser Asn 20 25 30 Leu Arg Arg Ser Asn Ser Ser Leu Phe Lys Ser Trp Arg Leu Gln Cys 35 40 45 Pro Phe Gly Asn Asn Asp Lys Gln Glu Ser Leu Ser Ser Trp Ile Pro 50 55 60 Glu Asn Ile Lys Lys Lys Glu Cys Val Tyr Phe Val Glu Ser Ser Lys 65 70 75 80 Leu Ser Asp Ala Gly Lys Val Val Cys Gln Cys Gly Tyr Thr His Glu 85 90 95 Gln His Leu Glu Glu Ala Thr Lys Pro His Thr Phe Gln Gly Thr Gln 100 105 110 Trp Asp Pro Lys Lys His Val Gln Glu Met Pro Thr Asp Ala Phe Gly 115 120 125 Asp Ile Val Phe Thr Gly Leu Ser Gln Lys Val Lys Lys Tyr Val Arg 130 135 140 Val Ser Gln Asp Thr Pro Ser Ser Val Ile Tyr His Leu Met Thr Gln 145 150 155 160 His Trp Gly Leu Asp Val Pro Asn Leu Leu Ile Ser Val Thr Gly Gly 165 170 175 Ala Lys Asn Phe Asn Met Lys Pro Arg Leu Lys Ser Ile Phe Arg Arg 180 185 190 Gly Leu Val Lys Val Ala Gln Thr Thr Gly Ala Trp Ile Ile Thr Gly 195 200 205 Gly Ser His Thr Gly Val Met Lys Gln Val Gly Glu Ala Val Arg Asp 210 215 220 Phe Ser Leu Ser Ser Ser Tyr Lys Glu Gly Glu Leu Ile Thr Ile Gly 225 230 235 240 Val Ala Thr Trp Gly Thr Val His Arg Arg Glu Gly Leu Ile His Pro 245 250 255 Thr Gly Ser Phe Pro Ala Glu Tyr Ile Leu Asp Glu Asp Gly Gln Gly 260 265 270 Asn Leu Thr Cys Leu Asp Ser Asn His Ser His Phe Ile Leu Val Asp 275 280 285 Asp Gly Thr His Gly Gln Tyr Gly Val Glu Ile Pro Leu Arg Thr Arg 290 295 300 Leu Glu Lys Phe Ile Ser Glu Gln Thr Lys Glu Arg Gly Gly Val Ala 305 310 315 320 Ile Lys Ile Pro Ile Val Cys Val Val Leu Glu Gly Gly Pro Gly Thr 325 330 335 Leu His Thr Ile Asp Asn Ala Thr Thr Asn Gly Thr Pro Cys Val Val 340 345 350 Val Glu Gly Ser Gly Arg Val Ala Asp Val Ile Ala Gln Val Ala Asn 355 360 365 Leu Pro Val Ser Asp Ile Thr Ile Ser Leu Ile Gln Gln Lys Leu Ser 370 375 380 Val Phe Phe Gln Glu Met Phe Glu Thr Phe Thr Glu Ser Arg Ile Val 385 390 395 400 Glu Trp Thr Lys Lys Ile Gln Asp Ile Val Arg Arg Arg Gln Leu Leu 405 410 415 Thr Val Phe Arg Glu Gly Lys Asp Gly Gln Gln Asp Val Asp Val Ala 420 425 430 Ile Leu Gln Ala Leu Leu Lys Ala Ser Arg Ser Gln Asp His Phe Gly 435 440 445 His Glu Asn Trp Asp His Gln Leu Lys Leu Ala Val Ala Trp Asn Arg 450 455 460 Val Asp Ile Ala Arg Ser Glu Ile Phe Met Asp Glu Trp Gln Trp Lys 465 470 475 480 Pro Ser Asp Leu His Pro Thr Met Thr Ala Ala Leu Ile Ser Asn Lys 485 490 495 Pro Glu Phe Val Lys Leu Phe Leu Glu Asn Gly Val Gln Leu Lys Glu 500 505 510 Phe Val Thr Trp Asp Thr Leu Leu Tyr Leu Tyr Glu Asn Leu Asp Pro 515 520 525 Ser Cys Leu Phe His Ser Lys Leu Gln Lys Val Leu Val Glu Asp Pro 530 535 540 Glu Arg Pro Ala Cys Ala Pro Ala Ala Pro Arg Leu Gln Met His His 545 550 555 560 Val Ala Gln Val Leu Arg Glu Leu Leu Gly Asp Phe Thr Gln Pro Leu 565 570 575 Tyr Pro Arg Pro Arg His Asn Asp Arg Leu Arg Leu Leu Leu Pro Val 580 585 590 Pro His Val Lys Leu Asn Val Gln Gly Val Ser Leu Arg Ser Leu Tyr 595 600 605 Lys Arg Ser Ser Gly His Val Thr Phe Thr Met Asp Pro Ile Arg Asp 610 615 620 Leu Leu Ile Trp Ala Ile Val Gln Asn Arg Arg Glu Leu Ala Gly Ile 625 630 635 640 Ile Trp Ala Gln Ser Gln Asp Cys Ile Ala Ala Ala Leu Ala Cys Ser 645 650 655 Lys Ile Leu Lys Glu Leu Ser Lys Glu Glu Glu Asp Thr Asp Ser Ser 660 665 670 Glu Glu Met Leu Ala Leu Ala Glu Glu Tyr Glu His Arg Ala Ile Gly 675 680 685 Val Phe Thr Glu Cys Tyr Arg Lys Asp Glu Glu Arg Ala Gln Lys Leu 690 695 700 Leu Thr Arg Val Ser Glu Ala Trp Gly Lys Thr Thr Cys Leu Gln Leu 705 710 715 720 Ala Leu Glu Ala Lys Asp Met Lys Phe Val Ser His Gly Gly Ile Gln 725 730 735 Ala Phe Leu Thr Lys Val Trp Trp Gly Gln Leu Ser Val Asp Asn Gly 740 745 750 Leu Trp Arg Val Thr Leu Cys Met Leu Ala Phe Pro Leu Leu Leu Thr 755 760 765 Gly Leu Ile Ser Phe Arg Glu Lys Arg Leu Gln Asp Val Gly Thr Pro 770 775 780 Ala Ala Arg Ala Arg Ala Phe Phe Thr Ala Pro Val Val Val Phe His 785 790 795 800 Leu Asn Ile Leu Ser Tyr Phe Ala Phe Leu Cys Leu Phe Ala Tyr Val 805 810 815 Leu Met Val Asp Phe Gln Pro Val Pro Ser Trp Cys Glu Cys Ala Ile 820 825 830 Tyr Leu Trp Leu Phe Ser Leu Val Cys Glu Glu Met Arg Gln Leu Phe 835 840 845 Tyr Asp Pro Asp Glu Cys Gly Leu Met Lys Lys Ala Ala Leu Tyr Phe 850 855 860 Ser Asp Phe Trp Asn Lys Leu Asp Val Gly Ala Ile Leu Leu Phe Val 865 870 875 880 Ala Gly Leu Thr Cys Arg Leu Ile Pro Ala Thr Leu Tyr Pro Gly Arg 885 890 895 Val Ile Leu Ser Leu Asp Phe Ile Leu Phe Cys Leu Arg Leu Met His 900 905 910 Ile Phe Thr Ile Ser Lys Thr Leu Gly Pro Lys Ile Ile Ile Val Lys 915 920 925 Arg Met Met Lys Asp Val Phe Phe Phe Leu Phe Leu Leu Ala Val Trp 930 935 940 Val Val Ser Phe Gly Val Ala Lys Gln Ala Ile Leu Ile His Asn Glu 945 950 955 960 Arg Arg Val Asp Trp Leu Phe Arg Gly Ala Val Tyr His Ser Tyr Leu 965 970 975 Thr Ile Phe Gly Gln Ile Pro Gly Tyr Ile Asp Gly Val Asn Phe Asn 980 985 990 Pro Glu His Cys Ser Pro Asn Gly Thr Asp Pro Tyr Lys Pro Lys Cys 995 1000 1005 Pro Glu Ser Asp Ala Thr Gln Gln Arg Pro Ala Phe Pro Glu Trp 1010 1015 1020 Leu Thr Val Leu Leu Leu Cys Leu Tyr Leu Leu Phe Thr Asn Ile 1025 1030 1035 Leu Leu Leu Asn Leu Leu Ile Ala Met Phe Asn Tyr Thr Phe Gln 1040 1045 1050 Gln Val Gln Glu His Thr Asp Gln Ile Trp Lys Phe Gln Arg His 1055 1060 1065 Asp Leu Ile Glu Glu Tyr His Gly Arg Pro Ala Ala Pro Pro Pro 1070 1075 1080 Phe Ile Leu Leu Ser His Leu Gln Leu Phe Ile Lys Arg Val Val 1085 1090 1095 Leu Lys Thr Pro Ala Lys Arg His Lys Gln Leu Lys Asn Lys Leu 1100 1105 1110 Glu Lys Asn Glu Glu Ala Ala Leu Leu Ser Trp Glu Ile Tyr Leu 1115 1120 1125 Lys Glu Asn Tyr Leu Gln Asn Arg Gln Phe Gln Gln Lys Gln Arg 1130 1135 1140 Pro Glu Gln Lys Ile Glu Asp Ile Ser Asn Lys Val Asp Ala Met 1145 1150 1155 Val Asp Leu Leu Asp Leu Asp Pro Leu Lys Arg Ser Gly Ser Met 1160 1165 1170 Glu Gln Arg Leu Ala Ser Leu Glu Glu Gln Val Ala Gln Thr Ala 1175 1180 1185 Arg Ala Leu His Trp Ile Val Arg Thr Leu Arg Ala Ser Gly Phe 1190 1195 1200 Ser Ser Glu Ala Asp Val Pro Thr Leu Ala Ser Gln Lys Ala Ala 1205 1210 1215 Glu Glu Pro Asp Ala Glu Pro Gly Gly Arg Lys Lys Thr Glu Glu 1220 1225 1230 Pro Gly Asp Ser Tyr His Val Asn Ala Arg His Leu Leu Tyr Pro 1235 1240 1245 Asn Cys Pro Val Thr Arg Phe Pro Val Pro Asn Glu Lys Val Pro 1250 1255 1260 Trp Glu Thr Glu Phe Leu Ile Tyr Asp Pro Pro Phe Tyr Thr Ala 1265 1270 1275 Glu Arg Lys Asp Ala Ala Ala Met Asp Pro Met Gly Asp Thr Leu 1280 1285 1290 Glu Pro Leu Ser Thr Ile Gln Tyr Asn Val Val Asp Gly Leu Arg 1295 1300 1305 Asp Arg Arg Ser Phe His Gly Pro Tyr Thr Val Gln Ala Gly Leu 1310 1315 1320 Pro Leu Asn Pro Met Gly Arg Thr Gly Leu Arg Gly Arg Gly Ser 1325 1330 1335 Leu Ser Cys Phe Gly Pro Asn His Thr Leu Tyr Pro Met Val Thr 1340 1345 1350 Arg Trp Arg Arg Asn Glu Asp Gly Ala Ile Cys Arg Lys Ser Ile 1355 1360 1365 Lys Lys Met Leu Glu Val Leu Val Val Lys Leu Pro Leu Ser Glu 1370 1375 1380 His Trp Ala Leu Pro Gly Gly Ser Arg Glu Pro Gly Glu Met Leu 1385 1390 1395 Pro Arg Lys Leu Lys Arg Ile Leu Arg Gln Glu His Trp Pro Ser 1400 1405 1410 Phe Glu Asn Leu Leu Lys Cys Gly Met Glu Val Tyr Lys Gly Tyr 1415 1420 1425 Met Asp Asp Pro Arg Asn Thr Asp Asn Ala Trp Ile Glu Thr Val 1430 1435 1440 Ala Val Ser Val His Phe Gln Asp Gln Asn Asp Val Glu Leu Asn 1445 1450 1455 Arg Leu Asn Ser Asn Leu His Ala Cys Asp Ser Gly Ala Ser Ile 1460 1465 1470 Arg Trp Gln Val Val Asp Arg Arg Ile Pro Leu Tyr Ala Asn His 1475 1480 1485 Lys Thr Leu Leu Gln Lys Ala Ala Ala Glu Phe Gly Ala His Tyr 1490 1495 1500 4 3754 DNA Homo sapiens 4 agacaaggag cttgagatca acctgagcaa catagggaga ccccatctct acaaataact 60 gaaaaaaaaa aaaagctagg tgtggtggta catacctgtg gtcccatcta ctcaggaggc 120 tgagacagga ggattgcgtg agcctggctc aagacaaggc gtgccggtcg tggggctggt 180 ggtggaaggc ggtcccaacg tcatcctgtc agtgtgggag actgtcaagg acaaggaccc 240 agtggtggtg tgtgagggca caggtagggc ggctgacctc ctggccttca cacacaaaca 300 cctggcagat gaagggatgc tgcgacctca ggtgaaagag gagatcatct gcatgattca 360 gaacactttc aactttagtc ttaaacagtc caagcacctt ttccaaattc taatggagtg 420 tatggttcac agggattgta ttaccatatt tgatgctgac tctgaagagc agcaagacct 480 ggacttagca atcctaacag ctttgctgaa gggcacaaat ttatcagcgt cagagcaatt 540 aaatctggca atggcttggg acagggtgga cattgccaag aaacatatcc taatttatga 600 acaacactgg aagcctgatg ccctggaaca agcaatgtca gatgctttag tgatggatcg 660 ggtggatttt gtgaagctct taatagaata tggagtgaac ctccatcgct ttcttaccat 720 ccctcgactg gaagagctct acaatacaaa acaaggacct actaatacac tcttgcatca 780 tctcgtccaa gatgtgaaac agcataccct tctttcaggc taccgaataa ccttgattga 840 cattggatta gtagtagaat acctcattgg tagagcatat cgcagcaact acactagaaa 900 acatttcaga gccctctaca acaacctcta cagaaaatac aagcaccaga gacactcctc 960 aggaaataga aatgagtctg cagaaagtac gctgcactcc cagttcatta gaactgcaca 1020 gccatacaaa ttcaaggaaa agtctatagt ccttcataaa tcaaggaaga agtcaaaaga 1080 acaaaatgta tcagatgacc ctgagtctac tggctttctt tacccttaca atgacctgct 1140 ggtttgggct gtgctgatga aaaggcagaa gatggctatg ttcttctggc agcatggaga 1200 ggaggccacg gttaaagccg tgattgcgtg tatcctctac cgggcaatgg cccatgaagc 1260 taaggagagt cacatggtgg atgatgcctc agaagagttg aagaattact caaaacagtt 1320 tggccagctg gctctggact tgttggagaa ggcattcaag cagaatgagc gcatggccat 1380 gacgctgttg acgtatgaac tcaggaactg gagcaattcg acctgcctta aactggccgt 1440 gtcgggagga ttacgaccct ttgtttcaca tacttgtacc cagatgctac tgacagacat 1500 gtggatgggg aggctgaaaa tgaggaaaaa ctcttggtta aagattatta taagcattat 1560 tttaccaccc accattttga cactggaatt taaaagcaaa gctgagatgt cacatgttcc 1620 ccagtcccag gacttccaat ttatgtggta ttacagtgac cagaacgcca gcagttccaa 1680 agaaagtgct tctgtgaaag agtatgattt ggaaaggggc catgatgaga aactggatga 1740 aaatcagcat tttggtttgg aaagtgggca ccaacacctt ccgtggacca ggaaagtcta 1800 tgagttctac agtgctccaa ttgtcaagtt ttggttttat acgatggcgt atttggcatt 1860 cctcatgctg ttcacttaca ccgtgttggt ggagatgcag ccccagccca gcgtgcagga 1920 gtggcttgtt agcatttaca tcttcaccaa tgctattgag gtggtcaggg agatctgtat 1980 ttcagaacct gggaagttta cccaaaaggt gaaggtatgg attagtgagt actggaactt 2040 aacagaaact gtggccattg gcctgttttc agctggcttc gtccttcgat ggggtgaccc 2100 tccttttcac acagcgggaa gactgatcta ctgcatagac atcatattct ggttctcacg 2160 gctcctggac ttctttgctg tgaatcaaca tgcaggtcca tatgtgacca tgattgcaaa 2220 aatgacagca aacatgttct atattgtgat catcatggcc atagtcctgc tgagctttgg 2280 agtggcacgc aaggccatcc tttcgccaaa agagccacca tcttggagtc tagctcgaga 2340 tattgtattt gagccatact ggatgatata cggagaagtc tatgctggag aaatagatgt 2400 ttgttcaagc cagccatcct gccctcctgg ttcttttctt actccattct tgcaagctgt 2460 ctacctcttc gtgcaatata tcatcatggt gaacctgttg attgctttct tcaacaacgt 2520 ttacttagat atggaatcca tttcaaataa cctgtggaaa tacaaccgct atcgctacat 2580 catgacctac cacgagaagc cctggctgcc cccacctctc atcctgctga gccacgtggg 2640 ccttctcctc cgccgcctgt gctgtcatcg agctcctcac gaccaggaag agggtgacgt 2700 tggattaaaa ctctacctca gtaaggagga tctgaaaaaa cttcatgatt ttgaggagca 2760 gtgcgtggaa aaatacttcc atgagaagat ggaagatgtg aattgtagtt gtgaggaacg 2820 aatccgagtg acatcagaaa gggttacaga gatgtacttc cagctgaaag aaatgaatga 2880 aaaggtgtct tttataaagg actccttact gtctttggac agccaggtgg gacacctgca 2940 ggatctctct gccctgactg tggataccct gaaagtcctt tctgctgttg acactttgca 3000 agaggatgag gctctcctgg ccaagagaaa gcattctact tgcaaaaaac ttccccacag 3060 ctggagcaat gtcatctgtg cagaggttct aggcagcatg gagatcgctg gagagaagaa 3120 ataccagtat tatagcatgc cctcttcttt gctgaggagc ctggctggag gccggcatcc 3180 cccaagagtg cagagggggg cacttcttga gattacaaac agtaaaagag aggctacaaa 3240 tgtaagaaat gaccaggaaa ggcaagaaac acaaagtagt atagtggttt ctggggtgtc 3300 tcctaacagg caagcacact caaagtatgg ccagtttctt ctggtcccct ctaatctaaa 3360 gcgagttcct ttttcagcag aaactgtctt gcctctgtcc agaccctctg tgccagatgt 3420 gctggcaact gaacaggaca tccagactga ggttcttgtt catctgactg ggcagacccc 3480 aattgtctct gactgggcat cagtggatga acccaaggaa aagcacgagc ctattgctca 3540 cttactggat ggacaagaca aggcagagca agtgctaccc actttgagtt gcacacctga 3600 acccatgaca atgagctccc ctctccgcag atacaggccc ttcgctagga gtcatagttt 3660 tagattccat aaggaggaga aattgatgaa gatctgtaag attaaaagta aggaataaac 3720 atttaaaata tcagcattaa aaaaaaaaaa aaaa 3754 5 599 DNA Homo sapiens misc_feature (3)..(4) n = a,t,g or c 5 gtngaaaata aatattacag tgtttataaa ttagcagttc ttaagtcagt tgaatcatca 60 actcgtcata ctcacacaat ccctgtgaac catacactcc attagaattt ggaaaaggtg 120 cttggactgt ttaagactaa agttgaaagt gttctgaatc atgcagatga tctcctcttt 180 cacctgaggt cgcagcatcc cttcatctgc caggtgtttg tgtgtgaagg ccaggaggtc 240 agccgcccta cctgtaccct cacacaccac cactgggtcc ttgtccttga cagtctccca 300 cactgacagg atgacgttgg gaccgccttc caccaccagc cccacgaccg gcacgccttg 360 tcttgagcgg cagtgtattt tctgcagaga gaggtacttc tccaggttcc ttctgagctt 420 catttcattt ccatacttgc ccacggtccc atcatcagac aggatgaagt gcgagtgcat 480 gctgttgagt gttgtgagct tgctgagggg gttatccaga gtctggtaca ggcacaccac 540 atcttttcca taaggtctct ctggttctca atgacacccc aaagagggat tccaactgt 599 6 443 DNA Homo sapiens 6 tttcaagttg aaaaacaagt aaatggtacc ttgaatttgt atggctgtga gttctaatga 60 actgggagtg cagcgtactt tctgcagact catttctatt tcctgaggag tgtctctggt 120 gctgggaaag gttttgaaca aagctggtca ctctctggca cgttattatt ttatttttaa 180 aataaaacaa aaggagggga gattatagtg gtaacaatat tttatttcta gacaataatg 240 aaaaccaaat ataatgttgt gtcctaccaa atatccatgc ctctgtttct ctccatgatt 300 agcacccaac aagagcctct aatgatctca ttgtgcattt tgttatttcc taacatctta 360 gagaacacac tcttttgttc cctattatat atctgcatac cttcactacg tggaaattcc 420 attagaatga atggatgttc tct 443 7 3754 DNA Homo sapiens 7 agacaaggag cttgagatca acctgagcaa catagggaga ccccatctct acaaataact 60 gaaaaaaaaa aaaagctagg tgtggtggta catacctgtg gtcccatcta ctcaggaggc 120 tgagacagga ggattgcgtg agcctggctc aagacaaggc gtgccggtcg tggggctggt 180 ggtggaaggc ggtcccaacg tcatcctgtc agtgtgggag actgtcaagg acaaggaccc 240 agtggtggtg tgtgagggca caggtagggc ggctgacctc ctggccttca cacacaaaca 300 cctggcagat gaagggatgc tgcgacctca ggtgaaagag gagatcatct gcatgattca 360 gaacactttc aactttagtc ttaaacagtc caagcacctt ttccaaattc taatggagtg 420 tatggttcac agggattgta ttaccatatt tgatgctgac tctgaagagc agcaagacct 480 ggacttagca atcctaacag ctttgctgaa gggcacaaat ttatcagcgt cagagcaatt 540 aaatctggca atggcttggg acagggtgga cattgccaag aaacatatcc taatttatga 600 acaacactgg aagcctgatg ccctggaaca agcaatgtca gatgctttag tgatggatcg 660 ggtggatttt gtgaagctct taatagaata tggagtgaac ctccatcgct ttcttaccat 720 ccctcgactg gaagagctct acaatacaaa acaaggacct actaatacac tcttgcatca 780 tctcgtccaa gatgtgaaac agcataccct tctttcaggc taccgaataa ccttgattga 840 cattggatta gtagtagaat acctcattgg tagagcatat cgcagcaact acactagaaa 900 acatttcaga gccctctaca acaacctcta cagaaaatac aagcaccaga gacactcctc 960 aggaaataga aatgagtctg cagaaagtac gctgcactcc cagttcatta gaactgcaca 1020 gccatacaaa ttcaaggaaa agtctatagt ccttcataaa tcaaggaaga agtcaaaaga 1080 acaaaatgta tcagatgacc ctgagtctac tggctttctt tacccttaca atgacctgct 1140 ggtttgggct gtgctgatga aaaggcagaa gatggctatg ttcttctggc agcatggaga 1200 ggaggccacg gttaaagccg tgattgcgtg tatcctctac cgggcaatgg cccatgaagc 1260 taaggagagt cacatggtgg atgatgcctc agaagagttg aagaattact caaaacagtt 1320 tggccagctg gctctggact tgttggagaa ggcattcaag cagaatgagc gcatggccat 1380 gacgctgttg acgtatgaac tcaggaactg gagcaattcg acctgcctta aactggccgt 1440 gtcgggagga ttacgaccct ttgtttcaca tacttgtacc cagatgctac tgacagacat 1500 gtggatgggg aggctgaaaa tgaggaaaaa ctcttggtta aagattatta taagcattat 1560 tttaccaccc accattttga cactggaatt taaaagcaaa gctgagatgt cacatgttcc 1620 ccagtcccag gacttccaat ttatgtggta ttacagtgac cagaacgcca gcagttccaa 1680 agaaagtgct tctgtgaaag agtatgattt ggaaaggggc catgatgaga aactggatga 1740 aaatcagcat tttggtttgg aaagtgggca ccaacacctt ccgtggacca ggaaagtcta 1800 tgagttctac agtgctccaa ttgtcaagtt ttggttttat acgatggcgt atttggcatt 1860 cctcatgctg ttcacttaca ccgtgttggt ggagatgcag ccccagccca gcgtgcagga 1920 gtggcttgtt agcatttaca tcttcaccaa tgctattgag gtggtcaggg agatctgtat 1980 ttcagaacct gggaagttta cccaaaaggt gaaggtatgg attagtgagt actggaactt 2040 aacagaaact gtggccattg gcctgttttc agctggcttc gtccttcgat ggggtgaccc 2100 tccttttcac acagcgggaa gactgatcta ctgcatagac atcatattct ggttctcacg 2160 gctcctggac ttctttgctg tgaatcaaca tgcaggtcca tatgtgacca tgattgcaaa 2220 aatgacagca aacatgttct atattgtgat catcatggcc atagtcctgc tgagctttgg 2280 agtggcacgc aaggccatcc tttcgccaaa agagccacca tcttggagtc tagctcgaga 2340 tattgtattt gagccatact ggatgatata cggagaagtc tatgctggag aaatagatgt 2400 ttgttcaagc cagccatcct gccctcctgg ttcttttctt actccattct tgcaagctgt 2460 ctacctcttc gtgcaatata tcatcatggt gaacctgttg attgctttct tcaacaacgt 2520 ttacttagat atggaatcca tttcaaataa cctgtggaaa tacaaccgct atcgctacat 2580 catgacctac cacgagaagc cctggctgcc cccacctctc atcctgctga gccacgtggg 2640 ccttctcctc cgccgcctgt gctgtcatcg agctcctcac gaccaggaag agggtgacgt 2700 tggattaaaa ctctacctca gtaaggagga tctgaaaaaa cttcatgatt ttgaggagca 2760 gtgcgtggaa aaatacttcc atgagaagat ggaagatgtg aattgtagtt gtgaggaacg 2820 aatccgagtg acatcagaaa gggttacaga gatgtacttc cagctgaaag aaatgaatga 2880 aaaggtgtct tttataaagg actccttact gtctttggac agccaggtgg gacacctgca 2940 ggatctctct gccctgactg tggataccct gaaagtcctt tctgctgttg acactttgca 3000 agaggatgag gctctcctgg ccaagagaaa gcattctact tgcaaaaaac ttccccacag 3060 ctggagcaat gtcatctgtg cagaggttct aggcagcatg gagatcgctg gagagaagaa 3120 ataccagtat tatagcatgc cctcttcttt gctgaggagc ctggctggag gccggcatcc 3180 cccaagagtg cagagggggg cacttcttga gattacaaac agtaaaagag aggctacaaa 3240 tgtaagaaat gaccaggaaa ggcaagaaac acaaagtagt atagtggttt ctggggtgtc 3300 tcctaacagg caagcacact caaagtatgg ccagtttctt ctggtcccct ctaatctaaa 3360 gcgagttcct ttttcagcag aaactgtctt gcctctgtcc agaccctctg tgccagatgt 3420 gctggcaact gaacaggaca tccagactga ggttcttgtt catctgactg ggcagacccc 3480 aattgtctct gactgggcat cagtggatga acccaaggaa aagcacgagc ctattgctca 3540 cttactggat ggacaagaca aggcagagca agtgctaccc actttgagtt gcacacctga 3600 acccatgaca atgagctccc ctctccgcag atacaggccc ttcgctagga gtcatagttt 3660 tagattccat aaggaggaga aattgatgaa gatctgtaag attaaaagta aggaataaac 3720 atttaaaata tcagcattaa aaaaaaaaaa aaaa 3754 8 3506 DNA Homo sapiens 8 agctaatata ttttgtttgg tggaagaaga attgtaaaca cgtggatagt atttgtattg 60 ggtaactgcc ggtaatgtca ggtgagccaa tcataaatgt tggaaaatat agactctgtt 120 acctttcaag acttcatatt aaaacattgt ccctctctta cttcatctta cagcaacgtt 180 tacttagata tggaatccat ttcaaataac ctgtggaaat acaaccgcta tcgctacatc 240 atgacctacc acgagaagcc ctggctgccc ccacctctca tcctgctgag ccacgtgggc 300 cttctcctcc gccgcctgtg ctgtcatcga gctcctcacg accaagaaga gggtgacgtt 360 ggattaaaac tctacctcag taaggaggat ctgaaaaaac ttcatgattt tgaggagcag 420 tgcgtggaaa aatacttcca tgagaagatg gaagatgtga attgtagttg tgaggaacga 480 atccgagtga catcagaaag ggttacagag atgtacttcc agctgaaaga aatgaatgaa 540 aaggtgtctt ttataaagga ctccttactg tctttggaca gccaggtggg acacctgcag 600 gatctctctg ccctgactgt ggataccctg aaagtccttt ctgctgttga cactttgcaa 660 gaggatgagg ctctcctggc caagagaaag cattctactt gaaaaaaact tccccacagc 720 tggagcaatg tcatctgtgc agaggttcta ggcagcatgg agatcgctgg agagaagaaa 780 taccagtatt atagcatgcc ctcttctttg ctgaggagcc tggctggagg ccggcatccc 840 ccaagagtgc agaggggggc acttcttgag attacaaaca gtaaaagaga ggctacaaat 900 gtaagaaatg accaggagag gcaagaaaca caaagtagta tagtggtttc tggggtgtct 960 cctaacaggc aagcacactc aaagtatggc cagtttcttc tggtcccctc taatctaaag 1020 cgagttcctt tttcagcaga aactgtcttg cctctgtcca gaccctctgt gccagatgtg 1080 ctggcaactg aacaggacat ccagactgag gttcttgttc atctgactgg gcagacccca 1140 gttgtctctg actgggcatc agtggatgaa cccaaggaaa agcacgagcc tattgctcac 1200 ttactggatg gacaagacaa ggtagagcaa gtgctaccca ctttgagttg cacacctgaa 1260 cccatgacaa tgagctcccc tctttcccaa gccaagatca tgcaaactgg aggtggatat 1320 gtaaactggg cattttcaga aggtgatgaa actggtgtgt ttagcatcaa gaaaaagtgg 1380 caaacctgct tgccctccac ttgtgacagt gattcctctc ggagtgaaca gcaccagaag 1440 caggcccagg acagctccct atctgataac tcaacaagat cggcccagag tagtgaatgc 1500 tcagaggtgg gaccatggct tcagccaaac acatcctttt ggatcaatcc tctccgcaga 1560 tacaggccct tcgctaggag tcatagtttt agattccata aggaggagaa attgatgaag 1620 atctgtaaga ttaaaaatct ttcaggctct tcagaaatag ggcagggagc atgggtcaaa 1680 gcgaaaatgc taaccaaaga caggagactg tcaaagaaaa agaagaatac tcaaggactc 1740 caggtgccaa tcataacagt caatgcctgc tctcagagtg accagttgaa tccagagcca 1800 ggagaaaaca gcatctctga agaggagtac agcaagaact ggttcacagt gtccaaattt 1860 agtcacacag gtgtagaacc ttacatacat cagaaaatga aaactaaaga aattggacaa 1920 tgtgctatac aaatcagtga ttacctaaag cagtctcagg aggatctcag caaaaactct 1980 ttgtggaatt ccaggagcac caacctcaat aggaactccc tgctgaaaag ttcaattgga 2040 gttgacaaga tctcagcctc cttaaaaagc cctcaagagc ctcaccatca ttattcagcc 2100 attgaaagga ataatttaat gaggctttct cagaccatac catttacacc agtccaactg 2160 tttgcaggag aagaaataac tgtctacagg ttggaggaga gttccccttt aaaccttgat 2220 aaaagcatgt cctcttggtc tcagcgtggg agagcggcaa tgatccaggt attgtcccga 2280 gaggagatgg atgggggcct ccgtaaagct atgagagtcg tcagcacttg gtctgaggat 2340 gacattctca agccgggaca agttttcatt gtcaagtcct ttcttcctga ggttgtgcgg 2400 acatggcata aaatcttcca ggagagcact gtgcttcatc tttgcctcag ggaaattcaa 2460 caacaaagag ctgctcaaaa attgatctat accttcaacc aagtgaaacc acaaaccata 2520 ccctacacac caaggttcct ggaagttttc ttaatctact gccattcagc caaccagtgg 2580 ttgaccattg agaagtatat gacaggggag ttccggaagt ataacaacaa caatggtgat 2640 gaaatcaccc ccaccaacac cctggaggag ctgatgttgg ctttctctca ctggacctat 2700 gagtacactc ggggagagcc gctggtttta gatttgcaag gtgttggaga aaatttgaca 2760 gatccatctg ttataaaacc tgaagtcaaa caatcaagag gaatggtgtt tggaccggcc 2820 aatttggggg aagatgcaat tagaaacttc attgcaaaac atcattgtaa ctcctgctgc 2880 cggaagctca aactcccgga tttaaaaaga aatgactatt cccctgaaag gataaattcc 2940 acctttggac ttgagataaa aatagaatca gctgaggagc ctccagcaag ggagacgggt 3000 agaaattccc cagaagatga tatgcaacta taaaaaggga ggagcaagaa gatcccagtg 3060 cttgccctgc ctgccaggaa ctctgtgata acatagattg atcaacgtga tgttgattac 3120 atcagcgtct ccttgggaca cgccttctga gcctcacatc tccttctgtt caaaggcctc 3180 attggtatat gatcaatggg ttctcctaga cactgacctc tgtccagggc actttgcagc 3240 tccatcctca agttccacac gaagatgctt ggatgagtca gctgggaata ttgttcttgt 3300 gtacctcatt gctttagctg gtcacttgga actttggagc agaatcctgc acattaaagg 3360 atggggttgg gggggataca tttattttat tttctcacta tgtatgcaga ctggaccccc 3420 tactactatt tgtcacctca cccacagatt gtatttatgt ctatatatat gttcataaaa 3480 agttatgtga taaaaaaaaa aaaaaa 3506 9 5397 DNA Homo sapiens 9 tcccagaaat cctggattaa aggagtattt gacaagagag aatgtagcac aatcataccc 60 agctcaaaaa atcctcacag atgtactcca gtatgccaag tctgccagaa tttaatcagg 120 tgttactgtg gccgactgat tggagaccat gctgggatag attattcctg gaccatctca 180 gctgccaagg gtaaagaaag tgaacaatgg tctgttgaaa agcacacaac gaaaagccca 240 acagatactt ttggcacgat taatttccaa gatggagagc acacccatca tgccaagtat 300 attagaactt cttatgatac aaaactggat catctgttac atttaatgtt gaaagagtgg 360 aaaatggaac tgcccaagct tgtgatctca gtccatgggg gcatccagaa ctttactatg 420 ccctctaaat ttaaagagat tttcagccaa ggtttggtta aagctgcaga gacaacagga 480 gcgtggataa taactgaagg catcaataca ggagtgtcca agcatgttgg ggatgccttg 540 aaatcccatt cctctcattc cttgagaaaa atctggacag ttggaatccc tccttggggt 600 gtcattgaga accagagaga ccttattgga aaagatgtgg tgtgcctgta ccagactctg 660 gataaccccc tcagcaagct cacaacactc aacagcatgc actcgcactt catcctgtct 720 gatgatggga ccgtgggcaa gtatggaaat gaaatgaagc tcagaaggaa cctggagaag 780 tacctctctc tgcagaaaat acactgccgc tcaagacaag gcgtgccggt cgtggggctg 840 gtggtggaag gcggtcccaa cgtcatcctg tcagtgtggg agactgtcaa ggacaaggac 900 ccagtggtgg tgtgtgaggg cacaggtagg gcggctgacc tcctggcctt cacacacaaa 960 cacctggcag atgaagggat gctgcgacct caggtgaaag aggagatcat ctgcatgatt 1020 cagaacactt tcaactttag tcttaaacag tccaagcacc ttttccaaat tctaatggag 1080 tgtatggttc acagggattg tattaccata tttgatgctg actctgaaga gcagcaagac 1140 ctggacttag caatcctaac agctttgctg aagggcacaa atttatcagc gtcagagcaa 1200 ttaaatctgg caatggcttg ggacagggtg gacattgcca agaaacatat cctaatttat 1260 gaacaacact ggaagcctga tgccctggaa caagcaatgt cagatgcttt agtgatggat 1320 cgggtggatt ttgtgaagct cttaatagaa tatggagtga acctccatcg ctttcttacc 1380 atccctcgac tggaagagct ctacaataca aaacaaggac ctactaatac actcttgcat 1440 catctcgtcc aagatgtgaa acagcatacc cttctttcag gctaccgaat aaccttgatt 1500 gacattggat tagtagtaga atacctcatt ggtagagcat atcgcagcaa ctacactaga 1560 aaacatttca gagccctcta caacaacctc tacagaaaat acaagagagt gaccagcttt 1620 gttcaaaacc tttcccagca ccagagacac tcctcaggaa atagaaatga gtctgcagaa 1680 agtacgctgc actcccagtt cattagaact gcacagccat acaaattcaa ggaaaagtct 1740 atagtccttc ataaatcaag gaagaagtca aaagaacaaa atgtatcaga tgaccctgag 1800 tctactggct ttctttaccc ttacaatgac ctgctggttt gggctgtgct gatgaaaagg 1860 cagaagatgg ctatgttctt ctggcagcat ggagaggagg ccacggttaa agccgtgatt 1920 gcgtgtatcc tctaccgggc aatggcccat gaagctaagg agagtcacat ggtggatgat 1980 gcctcagaag agttgaagaa ttactcaaaa cagtttggcc agctggctct ggacttgttg 2040 gagaaggcat tcaagcagaa tgagcgcatg gccatgacgc tgttgacgta tgaactcagg 2100 aactggagca attcgacctg ccttaaactg gccgtgtcgg gaggattacg accctttgtt 2160 tcacatactt gtacccagat gctactgaca gacatgtgga tggggaggct gaaaatgagg 2220 aaaaactctt ggttaaagat tattataagc attattttac cacccaccat tttgacactg 2280 gaatttaaaa gcaaagctga gatgtcacat gttccccagt cccaggactt ccaatttatg 2340 tggtattaca gtgaccagaa cgccagcagt tccaaagaaa agtatgattt ggaaaggggc 2400 catgatgaga aactggatga aaatcagcat tttggtttgg aaagtgggca ccaacacctt 2460 ccgtggacca ggaaagtcta tgagttctac agtgctccaa ttgtcaagtt ttggttttat 2520 acgatggcgt atttggcatt cctcatgctg ttcacttaca ccgtgttggt ggagatgcag 2580 ccccagccca gcgtgcagga gtggcttgtt agcatttaca tcttcaccaa tgctattgag 2640 gtggtcaggg agatctgtat ttcagaacct gggaagttta cccaaaaggt gaaggtatgg 2700 attagtgagt actggaactt aacagaaact gtggccattg gcctgttttc agctggcttc 2760 gtccttcgat ggggtgaccc tccttttcac acagcgggaa gactgatcta ctgcatagac 2820 atcatattct ggttctcacg gctcctggac ttctttgctg tgaatcaaca tgcaggtcca 2880 tatgtgacca tgattgcaaa aatgacagca aacatgttct atattgtgat catcatggcc 2940 atagtcctgc tgagctttgg agtggcacgc aaggccatcc tttcgccaaa agagccacca 3000 tcttggagtc tagctcgaga tattgtattt gagccatact ggatgatata cggagaagtc 3060 tatgctggag aaatagatgt ttgttcaagc cagccatcct gccctcctgg ttcttttctt 3120 actccattct tgcaagctgt ctacctcttc gtgcaatata tcatcatggt gaacctgttg 3180 attgctttct tcaacaacgt ttacttagat atggaatcca tttcaaataa cctgtggaaa 3240 tacaaccgct atcgctacat catgacctac cacgagaagc cctggctgcc cccacctctc 3300 atcctgctga gccacgtggg ccttctcctc cgccgcctgt gctgtcatcg agctcctcac 3360 gaccaagaag agggtgacgt tggattaaaa ctctacctca gtaaggagga tctgaaaaaa 3420 cttcatgatt ttgaggagca gtgcgtggaa aaatacttcc atgagaagat ggaagatgtg 3480 aattgtagtt gtgaggaacg aatccgagtg acatcagaaa gggttacaga gatgtacttc 3540 cagctgaaag aaatgaatga aaaggtgtct tttataaagg actccttact gtctttggac 3600 agccaggtgg gacacctgca ggatctctct gccctgactg tggataccct gaaagtcctt 3660 tctgctgttg acactttgca agaggatgag gctctcctgg ccaagagaaa gcattctact 3720 tgcaaaaaac ttccccacag ctggagcaat gtcatctgtg cagaggttct aggcagcatg 3780 gagatcgctg gagagaagaa ataccagtat tatagcatgc cctcttcttt gctgaggagc 3840 ctggctggag gccggcatcc cccaagagtg cagagggggg cacttcttga gattacaaac 3900 agtaaaagag aggctacaaa tgtaagaaat gaccaggaaa ggcaagaaac acaaagtagt 3960 atagtggttt ctggggtgtc tcctaacagg caagcacact caaagtatgg ccagtttctt 4020 ctggtcccct ctaatctaaa gcgagttcct ttttcagcag aaactgtctt gcctctgtcc 4080 agaccctctg tgccagatgt gctggcaact gaacaggaca tccagactga ggttcttgtt 4140 catctgactg ggcagacccc agttgtctct gactgggcat cagtggatga acccaaggaa 4200 aagcacgagc ctattgctca cttactggat ggacaagaca aggcagagca agtgctaccc 4260 actttgagtt gcacacctga acccatgaca atgagctccc ctctttccca agccaagatc 4320 atgcaaactg gaggtggata tgtaaactgg gcattttcag aaggtgatga aactggtgtg 4380 tttagcatca agaaaaagtg gcaaacctgc ttgccctcca cttgtgacag tgattcctct 4440 cggagtgaac agcaccagaa gcaggcccag gacagctccc tatctgataa ctcaacaaga 4500 tcggcccaga gtaagatctc agcctcctta aaaagccctc aagagcctca ccatcattat 4560 tcagccattg aaaggaataa tttaatgagg ctttctcaga ccataccatt tacaccagtc 4620 caactgtttg gagaagaaat aactgtctac aggttggagg agagttcccc tttaaacctt 4680 gataaaagca tgtcctcttg gtctcagcgt gggagagcgg caatgatcca ggtattgtcc 4740 cgagaggaga tggatggggg cctccgtaaa gctatgagag tcgtcagcac ttggtctgag 4800 gatgacattc tcaagccggg acaagttttc attgtcaagt cctttcttcc tgaggttgtg 4860 cggacatggc ataaaatctt ccaggagagc actgtgcttc atctttgcct cagggaaatt 4920 caacaacaaa gagctgctca aaaattgatc tataccttca accaagtgaa accacaaacc 4980 ataccctaca caccaaggtt cctggaagtt ttcttaatct actgccattc agccaaccag 5040 tggttgacca ttgagaagta tatgacaggg gagttccgga agtataacaa caacaatggt 5100 gatgaaatca cccccaccaa caccctggag gagctgatgt tggctttctc tcactggacc 5160 tatgagtaca ctcggggaga gctgctggtt ttagatttgc aaggtgttgg agaaaatttg 5220 acagatccat ctgttataaa acctgaagtc aaacaatcaa gaggaatggt gtttggaccg 5280 gccaatttgg gggaagatgc aattagaaac ttcattgcaa aacatcattg taactcctgc 5340 tgccggaagc tcaaactccc ggatttaaaa agaaatgact attcccctga aaggata 5397 10 6004 DNA Homo sapiens 10 actatttagt ggaaaatcag tcaacacact ttctggtagt gttggtggaa caggaacttg 60 gtttttagat atttttagcc tggatgctgt ttttctgaga ctgcccctct cggatcaagt 120 gtcagaacca acaaacattt ggttctctgt tgaactgaac atggaggcct ctcggaagtg 180 tgctggagag tatattgcaa aggaactatt acgaggttgg cagggtggga agtgttactg 240 tggccgactg attggagacc atgctgggat agattattcc tggaccatct cagctgccaa 300 gggtaaagaa agtgaacaat ggtctgttga aaagcacaca acgaaaagcc caacagatac 360 ttttggcacg attaatttcc aagatggaga gcacacccat catgccaagt atattagaac 420 ttcttatgat acaaaactgg atcatctgtt acatttaatg ttgaaagagt ggaaaatgga 480 actgcccaag cttgtgatct cagtccatgg gggcatccag aactttacta tgccctctaa 540 atttaaagag attttcagcc aaggtttggt taaagctgca gagacaacag gagcgtggat 600 aataactgaa ggcatcaata caggagtgtc caagcatgtt ggggatgcct tgaaatccca 660 ttcctctcat tccttgagaa aaatctggac agttggaatc cctccttggg gtgtcattga 720 gaaccagaga gaccttattg gaaaagatgt ggtgtgcctg taccagactc tggataaccc 780 cctcagcaag ctcacaacac tcaacagcat gcactcgcac ttcatcctgt ctgatgatgg 840 gaccgtgggc aagtatggaa atgaaatgaa gctcagaagg aacctggaga agtacctctc 900 tctgcagaaa atacactgcc gctcaagaca aggcgtgccg gtcgtggggc tggtggtgga 960 aggcggtccc aacgtcatcc tgtcagtgtg ggagactgtc aaggacaagg acccagtggt 1020 ggtgtgtgag ggcacaggta gggcggctga cctcctggcc ttcacacaca aacacctggc 1080 agatgaaggg atgctgcgac ctcaggtgaa agaggagatc atctgcatga ttcagaacac 1140 tttcaacttt agtcttaaac agtccaagca ccttttccaa attctaatgg agtgtatggt 1200 tcacagggat tgtattacca tatttgatgc tgactctgaa gagcagcaag acctggactt 1260 agcaatccta acagctttgc tgaagggcac aaatttatca gcgtcagagc aattaaatct 1320 ggcaatggct tgggacaggg tggacattgc caagaaacat atcctaattt atgaacaaca 1380 ctggaagcct gatgccctgg aacaagcaat gtcagatgct ttagtgatgg atcgggtgga 1440 ttttgtgaag ctcttaatag aatatggagt gaacctccat cgctttctta ccatccctcg 1500 actggaagag ctctacaata caaaacaagg acctactaat acactcttgc atcatctcgt 1560 ccaagatgtg aaacagcata cccttctttc aggctaccga ataaccttga ttgacattgg 1620 attagtagta gaatacctca ttggtagagc atatcgcagc aactacacta gaaaacattt 1680 cagagccctc tacaacaacc tctacagaaa atacaaggaa aagtctatag tccttcataa 1740 atcaaggaag aagtcaaaag aacaaaatgt atcagatgac cctgagtcta ctggctttct 1800 ttacccttac aatgacctgc tggtttgggc tgtgctgatg aaaaggcaga agatggctat 1860 gttcttctgg cagcatggag aggaggccac ggttaaagcc gtgattgcgt gtatcctcta 1920 ccgggcaatg gcccatgaag ctaaggagag tcacatggtg gatgatgcct cagaagagtt 1980 gaagaattac tcaaaacagt ttggccagct ggctctggac ttgttggaga aggcattcaa 2040 gcagaatgag cgcatggcca tgacgctgtt gacgtatgaa ctcaggaact ggagcaattc 2100 gacctgcctt aaactggccg tgtcgggagg attacgaccc tttgtttcac atacttgtac 2160 ccagatgcta ctgacagaca tgtggatggg gaggctgaaa atgaggaaaa actcttggtt 2220 aaagattatt ataagcatta ttttaccacc caccattttg acactggaat ttaaaagcaa 2280 agctgagatg tcacatgttc cccagtccca ggacttccaa tttatgtggt attacagtga 2340 ccagaacgcc agcagttcca aagaaagtgc ttctgtgaaa gagtatgatt tggaaagggg 2400 ccatgatgag aaactggatg aaaatcagca ttttggtttg gaaagtgggc accaacacct 2460 tccgtggacc aggaaagtct atgagttcta cagtgctcca attgtcaagt tttggtttta 2520 tacgatggcg tatttggcat tcctcatgct gttcacttac accgtgttgg tggagatgca 2580 gccccagccc agcgtgcagg agtggcttgt tagcatttac atcttcacca atgctattga 2640 ggtggtcagg gagatctgta tttcagaacc tgggaagttt acccaaaagg tgaaggtatg 2700 gattagtgag tactggaact taacagaaac tgtggccatt ggcctgtttt cagctggctt 2760 cgtccttcga tggggtgacc ctccttttca cacagcggga agactgatct actgcataga 2820 catcatattc tggttctcac ggctcctgga cttctttgct gtgaatcaac atgcaggtcc 2880 atatgtgacc atgattgcaa aaatgacagc aaacatgttc tatattgtga tcatcatggc 2940 catagtcctg ctgagctttg gagtggcacg caaggccatc ctttcgccaa aagagccacc 3000 atcttggagt ctagctcgag atattgtatt tgagccatac tggatgatat acggagaagt 3060 ctatgctgga gaaatagatg ggcgacccct tccttctgga gccttagctc tgaaaagccc 3120 ctggtggggg tgccctttca gatgccccct ttccatttca aaggctctga ttctcgatct 3180 tgaagccaaa tgcagcaccg acactcggct tcagtttcca ctgggacagc tggaggtctc 3240 ctttctagcc ccagcccagg cggccaagcc catcctggca tcagaacatg ctgagcggca 3300 ttttgtaggc aacgtttact tagatatgga atccatttca aataacctgt ggaaatacaa 3360 ccgctatcgc tacatcatga cctaccacga gaagccctgg ctgcccccac ctctcatcct 3420 gctgagccac gtgggccttc tcctccgccg cctgtgctgt catcgagctc ctcacgacca 3480 agaagagggt gacgttggat taaaactcta cctcagtaag gaggatctga aaaaacttca 3540 tgattttgag gagcagtgcg tggaaaaata cttccatgag aagatggaag atgtgaattg 3600 tagttgtgag gaacgaatcc gagtgacatc agaaagtgga gttggcggct gcttcactga 3660 agtgggtgaa gacagaatag gagaagcaga gactgcaatt acaggtggct tttgggagaa 3720 gtattacggt gcagggaagc ttggtatggg gacacgggtt acagagatgt acttccagct 3780 gaaagaaatg aatgaaaagg tgtcttttat aaaggactcc ttactgtctt tggacagcca 3840 ggtgggacac ctgcaggatc tctctgccct gactgtggat accctgaaag tcctttctgc 3900 tgttgacact ttgcaagagg atgaggctct cctggccaag agaaagcatt ctacttgcaa 3960 aaaacttccc cacagctgga gcaatgtcat ctgtgcagag gttctaggca gcatggagat 4020 cgctggagag aagaaatacc agtattatag catgccctct tctttgctga ggagcctggc 4080 tggaggccgg catcccccaa gagtgcagag gggggcactt cttgagatta caaacagtaa 4140 aagagaggct acaaatgtaa gaaatgacca ggaaaggcaa gaaacacaaa gtagtatagt 4200 ggtttctggg gtgtctccta acaggcaagc acactcaaag tatggccagt ttcttctggt 4260 cccctctaat ctaaagcgag ttcctttttc agcagaaact gtcttgcctc tgtccagacc 4320 ctctgtgcca gatgtgctgg caactgaaca ggacatccag actgaggttc ttgttcatct 4380 gactgggcag accccagttg tctctgactg ggcatcagtg gatgaaccca aggaaaagca 4440 cgagcctatt gctcacttac tggatggaca agacaaggca gagcaagtgc tacccacttt 4500 gagttgcaca cctgaaccca tgacaatgag ctcccctctt tcccaagcca agatcatgca 4560 aactggaggt ggatatgtaa actgggcatt ttcagaaggt gatgaaactg gtgtgtttag 4620 catcaagaaa aagtggcaaa cctgcttgcc ctccacttgt gacagtgatt cctctcggag 4680 tgaacagcac cagaagcagg cccaggacag ctccctatct gataactcaa caagatcggc 4740 ccagagtagt gaatgctcag aggtgggacc atggcttcag ccaaacacat ccttttggat 4800 caatcctctc cgcagataca ggcccttcgc taggagtcat agttttagat tccataagga 4860 ggagaaattg atgaagatct gtaagattaa aaatctttca ggctcttcag aaatagggca 4920 gggagcatgg gtcaaagcga aaatgctaac caaagacagg agactgtcaa agaaaaagaa 4980 gaatactcaa ggactccagg tgccaatcat aacagtcaat gcctgctctc agagtgacca 5040 gttgaatcca gagccaggag aaaacagcat ctctgaagag gagtacagca agaactggtt 5100 cacagtgtcc aaatttagtc acacaggtgt agaaccttac atacatcaga aaatgaaaac 5160 taaagaaatt ggacaatgtg ctatacaaat cagtgattac ctaaagcagt ctcaagagga 5220 aaatgcagat cactcgcttc taattaaaat cggtcttcgg ctctctctgg aaaaagaaga 5280 tgaactgtgc aaagtgaaca caggagaaga aataactgtc tacaggttgg aggagagttc 5340 ccctttaaac cttgataaaa gcatgtcctc ttggtctcag cgtgggagag cggcaatgat 5400 ccaggtattg tcccgagagg agatggatgg gggcctccgt aaagctatga gagtcgtcag 5460 cacttggtct gaggatgaca ttctcaagcc gggacaagtt ttcattgtca agtcctttct 5520 tcctgaggtt gtgcggacat ggcataaaat cttccaggag agcactgtgc ttcatctttg 5580 cctcagggaa attcaacaac aaagagctgc tcaaaaattg atctatacct tcaaccaagt 5640 gaaaccacaa accataccct acacaccaag gttcctggaa gttttcttaa tctactgcca 5700 ttcagccaac cagtggttga ccattgagaa gtatatgaca ggggagttcc ggaagtataa 5760 caacaacaat ggtgatgaaa tcacccccac caacaccctg gaggagctga tgttggcttt 5820 ctctcactgg acctatgagt acactcgggg agagctgctg gttttagatt tgcaagtctt 5880 tggagagata atgcacattg tctgggtggt ggaagtggag acagaggctc accagcagca 5940 gttaggagga ggagctctgg agttgcaatg ccggagtctg acttctggtt ccactgtgaa 6000 ttag 6004 11 1799 PRT Homo sapiens 11 Ser Gln Lys Ser Trp Ile Lys Gly Val Phe Asp Lys Arg Glu Cys Ser 1 5 10 15 Thr Ile Ile Pro Ser Ser Lys Asn Pro His Arg Cys Thr Pro Val Cys 20 25 30 Gln Val Cys Gln Asn Leu Ile Arg Cys Tyr Cys Gly Arg Leu Ile Gly 35 40 45 Asp His Ala Gly Ile Asp Tyr Ser Trp Thr Ile Ser Ala Ala Lys Gly 50 55 60 Lys Glu Ser Glu Gln Trp Ser Val Glu Lys His Thr Thr Lys Ser Pro 65 70 75 80 Thr Asp Thr Phe Gly Thr Ile Asn Phe Gln Asp Gly Glu His Thr His 85 90 95 His Ala Lys Tyr Ile Arg Thr Ser Tyr Asp Thr Lys Leu Asp His Leu 100 105 110 Leu His Leu Met Leu Lys Glu Trp Lys Met Glu Leu Pro Lys Leu Val 115 120 125 Ile Ser Val His Gly Gly Ile Gln Asn Phe Thr Met Pro Ser Lys Phe 130 135 140 Lys Glu Ile Phe Ser Gln Gly Leu Val Lys Ala Ala Glu Thr Thr Gly 145 150 155 160 Ala Trp Ile Ile Thr Glu Gly Ile Asn Thr Gly Val Ser Lys His Val 165 170 175 Gly Asp Ala Leu Lys Ser His Ser Ser His Ser Leu Arg Lys Ile Trp 180 185 190 Thr Val Gly Ile Pro Pro Trp Gly Val Ile Glu Asn Gln Arg Asp Leu 195 200 205 Ile Gly Lys Asp Val Val Cys Leu Tyr Gln Thr Leu Asp Asn Pro Leu 210 215 220 Ser Lys Leu Thr Thr Leu Asn Ser Met His Ser His Phe Ile Leu Ser 225 230 235 240 Asp Asp Gly Thr Val Gly Lys Tyr Gly Asn Glu Met Lys Leu Arg Arg 245 250 255 Asn Leu Glu Lys Tyr Leu Ser Leu Gln Lys Ile His Cys Arg Ser Arg 260 265 270 Gln Gly Val Pro Val Val Gly Leu Val Val Glu Gly Gly Pro Asn Val 275 280 285 Ile Leu Ser Val Trp Glu Thr Val Lys Asp Lys Asp Pro Val Val Val 290 295 300 Cys Glu Gly Thr Gly Arg Ala Ala Asp Leu Leu Ala Phe Thr His Lys 305 310 315 320 His Leu Ala Asp Glu Gly Met Leu Arg Pro Gln Val Lys Glu Glu Ile 325 330 335 Ile Cys Met Ile Gln Asn Thr Phe Asn Phe Ser Leu Lys Gln Ser Lys 340 345 350 His Leu Phe Gln Ile Leu Met Glu Cys Met Val His Arg Asp Cys Ile 355 360 365 Thr Ile Phe Asp Ala Asp Ser Glu Glu Gln Gln Asp Leu Asp Leu Ala 370 375 380 Ile Leu Thr Ala Leu Leu Lys Gly Thr Asn Leu Ser Ala Ser Glu Gln 385 390 395 400 Leu Asn Leu Ala Met Ala Trp Asp Arg Val Asp Ile Ala Lys Lys His 405 410 415 Ile Leu Ile Tyr Glu Gln His Trp Lys Pro Asp Ala Leu Glu Gln Ala 420 425 430 Met Ser Asp Ala Leu Val Met Asp Arg Val Asp Phe Val Lys Leu Leu 435 440 445 Ile Glu Tyr Gly Val Asn Leu His Arg Phe Leu Thr Ile Pro Arg Leu 450 455 460 Glu Glu Leu Tyr Asn Thr Lys Gln Gly Pro Thr Asn Thr Leu Leu His 465 470 475 480 His Leu Val Gln Asp Val Lys Gln His Thr Leu Leu Ser Gly Tyr Arg 485 490 495 Ile Thr Leu Ile Asp Ile Gly Leu Val Val Glu Tyr Leu Ile Gly Arg 500 505 510 Ala Tyr Arg Ser Asn Tyr Thr Arg Lys His Phe Arg Ala Leu Tyr Asn 515 520 525 Asn Leu Tyr Arg Lys Tyr Lys Arg Val Thr Ser Phe Val Gln Asn Leu 530 535 540 Ser Gln His Gln Arg His Ser Ser Gly Asn Arg Asn Glu Ser Ala Glu 545 550 555 560 Ser Thr Leu His Ser Gln Phe Ile Arg Thr Ala Gln Pro Tyr Lys Phe 565 570 575 Lys Glu Lys Ser Ile Val Leu His Lys Ser Arg Lys Lys Ser Lys Glu 580 585 590 Gln Asn Val Ser Asp Asp Pro Glu Ser Thr Gly Phe Leu Tyr Pro Tyr 595 600 605 Asn Asp Leu Leu Val Trp Ala Val Leu Met Lys Arg Gln Lys Met Ala 610 615 620 Met Phe Phe Trp Gln His Gly Glu Glu Ala Thr Val Lys Ala Val Ile 625 630 635 640 Ala Cys Ile Leu Tyr Arg Ala Met Ala His Glu Ala Lys Glu Ser His 645 650 655 Met Val Asp Asp Ala Ser Glu Glu Leu Lys Asn Tyr Ser Lys Gln Phe 660 665 670 Gly Gln Leu Ala Leu Asp Leu Leu Glu Lys Ala Phe Lys Gln Asn Glu 675 680 685 Arg Met Ala Met Thr Leu Leu Thr Tyr Glu Leu Arg Asn Trp Ser Asn 690 695 700 Ser Thr Cys Leu Lys Leu Ala Val Ser Gly Gly Leu Arg Pro Phe Val 705 710 715 720 Ser His Thr Cys Thr Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg 725 730 735 Leu Lys Met Arg Lys Asn Ser Trp Leu Lys Ile Ile Ile Ser Ile Ile 740 745 750 Leu Pro Pro Thr Ile Leu Thr Leu Glu Phe Lys Ser Lys Ala Glu Met 755 760 765 Ser His Val Pro Gln Ser Gln Asp Phe Gln Phe Met Trp Tyr Tyr Ser 770 775 780 Asp Gln Asn Ala Ser Ser Ser Lys Glu Lys Tyr Asp Leu Glu Arg Gly 785 790 795 800 His Asp Glu Lys Leu Asp Glu Asn Gln His Phe Gly Leu Glu Ser Gly 805 810 815 His Gln His Leu Pro Trp Thr Arg Lys Val Tyr Glu Phe Tyr Ser Ala 820 825 830 Pro Ile Val Lys Phe Trp Phe Tyr Thr Met Ala Tyr Leu Ala Phe Leu 835 840 845 Met Leu Phe Thr Tyr Thr Val Leu Val Glu Met Gln Pro Gln Pro Ser 850 855 860 Val Gln Glu Trp Leu Val Ser Ile Tyr Ile Phe Thr Asn Ala Ile Glu 865 870 875 880 Val Val Arg Glu Ile Cys Ile Ser Glu Pro Gly Lys Phe Thr Gln Lys 885 890 895 Val Lys Val Trp Ile Ser Glu Tyr Trp Asn Leu Thr Glu Thr Val Ala 900 905 910 Ile Gly Leu Phe Ser Ala Gly Phe Val Leu Arg Trp Gly Asp Pro Pro 915 920 925 Phe His Thr Ala Gly Arg Leu Ile Tyr Cys Ile Asp Ile Ile Phe Trp 930 935 940 Phe Ser Arg Leu Leu Asp Phe Phe Ala Val Asn Gln His Ala Gly Pro 945 950 955 960 Tyr Val Thr Met Ile Ala Lys Met Thr Ala Asn Met Phe Tyr Ile Val 965 970 975 Ile Ile Met Ala Ile Val Leu Leu Ser Phe Gly Val Ala Arg Lys Ala 980 985 990 Ile Leu Ser Pro Lys Glu Pro Pro Ser Trp Ser Leu Ala Arg Asp Ile 995 1000 1005 Val Phe Glu Pro Tyr Trp Met Ile Tyr Gly Glu Val Tyr Ala Gly 1010 1015 1020 Glu Ile Asp Val Cys Ser Ser Gln Pro Ser Cys Pro Pro Gly Ser 1025 1030 1035 Phe Leu Thr Pro Phe Leu Gln Ala Val Tyr Leu Phe Val Gln Tyr 1040 1045 1050 Ile Ile Met Val Asn Leu Leu Ile Ala Phe Phe Asn Asn Val Tyr 1055 1060 1065 Leu Asp Met Glu Ser Ile Ser Asn Asn Leu Trp Lys Tyr Asn Arg 1070 1075 1080 Tyr Arg Tyr Ile Met Thr Tyr His Glu Lys Pro Trp Leu Pro Pro 1085 1090 1095 Pro Leu Ile Leu Leu Ser His Val Gly Leu Leu Leu Arg Arg Leu 1100 1105 1110 Cys Cys His Arg Ala Pro His Asp Gln Glu Glu Gly Asp Val Gly 1115 1120 1125 Leu Lys Leu Tyr Leu Ser Lys Glu Asp Leu Lys Lys Leu His Asp 1130 1135 1140 Phe Glu Glu Gln Cys Val Glu Lys Tyr Phe His Glu Lys Met Glu 1145 1150 1155 Asp Val Asn Cys Ser Cys Glu Glu Arg Ile Arg Val Thr Ser Glu 1160 1165 1170 Arg Val Thr Glu Met Tyr Phe Gln Leu Lys Glu Met Asn Glu Lys 1175 1180 1185 Val Ser Phe Ile Lys Asp Ser Leu Leu Ser Leu Asp Ser Gln Val 1190 1195 1200 Gly His Leu Gln Asp Leu Ser Ala Leu Thr Val Asp Thr Leu Lys 1205 1210 1215 Val Leu Ser Ala Val Asp Thr Leu Gln Glu Asp Glu Ala Leu Leu 1220 1225 1230 Ala Lys Arg Lys His Ser Thr Cys Lys Lys Leu Pro His Ser Trp 1235 1240 1245 Ser Asn Val Ile Cys Ala Glu Val Leu Gly Ser Met Glu Ile Ala 1250 1255 1260 Gly Glu Lys Lys Tyr Gln Tyr Tyr Ser Met Pro Ser Ser Leu Leu 1265 1270 1275 Arg Ser Leu Ala Gly Gly Arg His Pro Pro Arg Val Gln Arg Gly 1280 1285 1290 Ala Leu Leu Glu Ile Thr Asn Ser Lys Arg Glu Ala Thr Asn Val 1295 1300 1305 Arg Asn Asp Gln Glu Arg Gln Glu Thr Gln Ser Ser Ile Val Val 1310 1315 1320 Ser Gly Val Ser Pro Asn Arg Gln Ala His Ser Lys Tyr Gly Gln 1325 1330 1335 Phe Leu Leu Val Pro Ser Asn Leu Lys Arg Val Pro Phe Ser Ala 1340 1345 1350 Glu Thr Val Leu Pro Leu Ser Arg Pro Ser Val Pro Asp Val Leu 1355 1360 1365 Ala Thr Glu Gln Asp Ile Gln Thr Glu Val Leu Val His Leu Thr 1370 1375 1380 Gly Gln Thr Pro Val Val Ser Asp Trp Ala Ser Val Asp Glu Pro 1385 1390 1395 Lys Glu Lys His Glu Pro Ile Ala His Leu Leu Asp Gly Gln Asp 1400 1405 1410 Lys Ala Glu Gln Val Leu Pro Thr Leu Ser Cys Thr Pro Glu Pro 1415 1420 1425 Met Thr Met Ser Ser Pro Leu Ser Gln Ala Lys Ile Met Gln Thr 1430 1435 1440 Gly Gly Gly Tyr Val Asn Trp Ala Phe Ser Glu Gly Asp Glu Thr 1445 1450 1455 Gly Val Phe Ser Ile Lys Lys Lys Trp Gln Thr Cys Leu Pro Ser 1460 1465 1470 Thr Cys Asp Ser Asp Ser Ser Arg Ser Glu Gln His Gln Lys Gln 1475 1480 1485 Ala Gln Asp Ser Ser Leu Ser Asp Asn Ser Thr Arg Ser Ala Gln 1490 1495 1500 Ser Lys Ile Ser Ala Ser Leu Lys Ser Pro Gln Glu Pro His His 1505 1510 1515 His Tyr Ser Ala Ile Glu Arg Asn Asn Leu Met Arg Leu Ser Gln 1520 1525 1530 Thr Ile Pro Phe Thr Pro Val Gln Leu Phe Gly Glu Glu Ile Thr 1535 1540 1545 Val Tyr Arg Leu Glu Glu Ser Ser Pro Leu Asn Leu Asp Lys Ser 1550 1555 1560 Met Ser Ser Trp Ser Gln Arg Gly Arg Ala Ala Met Ile Gln Val 1565 1570 1575 Leu Ser Arg Glu Glu Met Asp Gly Gly Leu Arg Lys Ala Met Arg 1580 1585 1590 Val Val Ser Thr Trp Ser Glu Asp Asp Ile Leu Lys Pro Gly Gln 1595 1600 1605 Val Phe Ile Val Lys Ser Phe Leu Pro Glu Val Val Arg Thr Trp 1610 1615 1620 His Lys Ile Phe Gln Glu Ser Thr Val Leu His Leu Cys Leu Arg 1625 1630 1635 Glu Ile Gln Gln Gln Arg Ala Ala Gln Lys Leu Ile Tyr Thr Phe 1640 1645 1650 Asn Gln Val Lys Pro Gln Thr Ile Pro Tyr Thr Pro Arg Phe Leu 1655 1660 1665 Glu Val Phe Leu Ile Tyr Cys His Ser Ala Asn Gln Trp Leu Thr 1670 1675 1680 Ile Glu Lys Tyr Met Thr Gly Glu Phe Arg Lys Tyr Asn Asn Asn 1685 1690 1695 Asn Gly Asp Glu Ile Thr Pro Thr Asn Thr Leu Glu Glu Leu Met 1700 1705 1710 Leu Ala Phe Ser His Trp Thr Tyr Glu Tyr Thr Arg Gly Glu Leu 1715 1720 1725 Leu Val Leu Asp Leu Gln Gly Val Gly Glu Asn Leu Thr Asp Pro 1730 1735 1740 Ser Val Ile Lys Pro Glu Val Lys Gln Ser Arg Gly Met Val Phe 1745 1750 1755 Gly Pro Ala Asn Leu Gly Glu Asp Ala Ile Arg Asn Phe Ile Ala 1760 1765 1770 Lys His His Cys Asn Ser Cys Cys Arg Lys Leu Lys Leu Pro Asp 1775 1780 1785 Leu Lys Arg Asn Asp Tyr Ser Pro Glu Arg Ile 1790 1795 12 2000 PRT Homo sapiens 12 Leu Phe Ser Gly Lys Ser Val Asn Thr Leu Ser Gly Ser Val Gly Gly 1 5 10 15 Thr Gly Thr Trp Phe Leu Asp Ile Phe Ser Leu Asp Ala Val Phe Leu 20 25 30 Arg Leu Pro Leu Ser Asp Gln Val Ser Glu Pro Thr Asn Ile Trp Phe 35 40 45 Ser Val Glu Leu Asn Met Glu Ala Ser Arg Lys Cys Ala Gly Glu Tyr 50 55 60 Ile Ala Lys Glu Leu Leu Arg Gly Trp Gln Gly Gly Lys Cys Tyr Cys 65 70 75 80 Gly Arg Leu Ile Gly Asp His Ala Gly Ile Asp Tyr Ser Trp Thr Ile 85 90 95 Ser Ala Ala Lys Gly Lys Glu Ser Glu Gln Trp Ser Val Glu Lys His 100 105 110 Thr Thr Lys Ser Pro Thr Asp Thr Phe Gly Thr Ile Asn Phe Gln Asp 115 120 125 Gly Glu His Thr His His Ala Lys Tyr Ile Arg Thr Ser Tyr Asp Thr 130 135 140 Lys Leu Asp His Leu Leu His Leu Met Leu Lys Glu Trp Lys Met Glu 145 150 155 160 Leu Pro Lys Leu Val Ile Ser Val His Gly Gly Ile Gln Asn Phe Thr 165 170 175 Met Pro Ser Lys Phe Lys Glu Ile Phe Ser Gln Gly Leu Val Lys Ala 180 185 190 Ala Glu Thr Thr Gly Ala Trp Ile Ile Thr Glu Gly Ile Asn Thr Gly 195 200 205 Val Ser Lys His Val Gly Asp Ala Leu Lys Ser His Ser Ser His Ser 210 215 220 Leu Arg Lys Ile Trp Thr Val Gly Ile Pro Pro Trp Gly Val Ile Glu 225 230 235 240 Asn Gln Arg Asp Leu Ile Gly Lys Asp Val Val Cys Leu Tyr Gln Thr 245 250 255 Leu Asp Asn Pro Leu Ser Lys Leu Thr Thr Leu Asn Ser Met His Ser 260 265 270 His Phe Ile Leu Ser Asp Asp Gly Thr Val Gly Lys Tyr Gly Asn Glu 275 280 285 Met Lys Leu Arg Arg Asn Leu Glu Lys Tyr Leu Ser Leu Gln Lys Ile 290 295 300 His Cys Arg Ser Arg Gln Gly Val Pro Val Val Gly Leu Val Val Glu 305 310 315 320 Gly Gly Pro Asn Val Ile Leu Ser Val Trp Glu Thr Val Lys Asp Lys 325 330 335 Asp Pro Val Val Val Cys Glu Gly Thr Gly Arg Ala Ala Asp Leu Leu 340 345 350 Ala Phe Thr His Lys His Leu Ala Asp Glu Gly Met Leu Arg Pro Gln 355 360 365 Val Lys Glu Glu Ile Ile Cys Met Ile Gln Asn Thr Phe Asn Phe Ser 370 375 380 Leu Lys Gln Ser Lys His Leu Phe Gln Ile Leu Met Glu Cys Met Val 385 390 395 400 His Arg Asp Cys Ile Thr Ile Phe Asp Ala Asp Ser Glu Glu Gln Gln 405 410 415 Asp Leu Asp Leu Ala Ile Leu Thr Ala Leu Leu Lys Gly Thr Asn Leu 420 425 430 Ser Ala Ser Glu Gln Leu Asn Leu Ala Met Ala Trp Asp Arg Val Asp 435 440 445 Ile Ala Lys Lys His Ile Leu Ile Tyr Glu Gln His Trp Lys Pro Asp 450 455 460 Ala Leu Glu Gln Ala Met Ser Asp Ala Leu Val Met Asp Arg Val Asp 465 470 475 480 Phe Val Lys Leu Leu Ile Glu Tyr Gly Val Asn Leu His Arg Phe Leu 485 490 495 Thr Ile Pro Arg Leu Glu Glu Leu Tyr Asn Thr Lys Gln Gly Pro Thr 500 505 510 Asn Thr Leu Leu His His Leu Val Gln Asp Val Lys Gln His Thr Leu 515 520 525 Leu Ser Gly Tyr Arg Ile Thr Leu Ile Asp Ile Gly Leu Val Val Glu 530 535 540 Tyr Leu Ile Gly Arg Ala Tyr Arg Ser Asn Tyr Thr Arg Lys His Phe 545 550 555 560 Arg Ala Leu Tyr Asn Asn Leu Tyr Arg Lys Tyr Lys Glu Lys Ser Ile 565 570 575 Val Leu His Lys Ser Arg Lys Lys Ser Lys Glu Gln Asn Val Ser Asp 580 585 590 Asp Pro Glu Ser Thr Gly Phe Leu Tyr Pro Tyr Asn Asp Leu Leu Val 595 600 605 Trp Ala Val Leu Met Lys Arg Gln Lys Met Ala Met Phe Phe Trp Gln 610 615 620 His Gly Glu Glu Ala Thr Val Lys Ala Val Ile Ala Cys Ile Leu Tyr 625 630 635 640 Arg Ala Met Ala His Glu Ala Lys Glu Ser His Met Val Asp Asp Ala 645 650 655 Ser Glu Glu Leu Lys Asn Tyr Ser Lys Gln Phe Gly Gln Leu Ala Leu 660 665 670 Asp Leu Leu Glu Lys Ala Phe Lys Gln Asn Glu Arg Met Ala Met Thr 675 680 685 Leu Leu Thr Tyr Glu Leu Arg Asn Trp Ser Asn Ser Thr Cys Leu Lys 690 695 700 Leu Ala Val Ser Gly Gly Leu Arg Pro Phe Val Ser His Thr Cys Thr 705 710 715 720 Gln Met Leu Leu Thr Asp Met Trp Met Gly Arg Leu Lys Met Arg Lys 725 730 735 Asn Ser Trp Leu Lys Ile Ile Ile Ser Ile Ile Leu Pro Pro Thr Ile 740 745 750 Leu Thr Leu Glu Phe Lys Ser Lys Ala Glu Met Ser His Val Pro Gln 755 760 765 Ser Gln Asp Phe Gln Phe Met Trp Tyr Tyr Ser Asp Gln Asn Ala Ser 770 775 780 Ser Ser Lys Glu Ser Ala Ser Val Lys Glu Tyr Asp Leu Glu Arg Gly 785 790 795 800 His Asp Glu Lys Leu Asp Glu Asn Gln His Phe Gly Leu Glu Ser Gly 805 810 815 His Gln His Leu Pro Trp Thr Arg Lys Val Tyr Glu Phe Tyr Ser Ala 820 825 830 Pro Ile Val Lys Phe Trp Phe Tyr Thr Met Ala Tyr Leu Ala Phe Leu 835 840 845 Met Leu Phe Thr Tyr Thr Val Leu Val Glu Met Gln Pro Gln Pro Ser 850 855 860 Val Gln Glu Trp Leu Val Ser Ile Tyr Ile Phe Thr Asn Ala Ile Glu 865 870 875 880 Val Val Arg Glu Ile Cys Ile Ser Glu Pro Gly Lys Phe Thr Gln Lys 885 890 895 Val Lys Val Trp Ile Ser Glu Tyr Trp Asn Leu Thr Glu Thr Val Ala 900 905 910 Ile Gly Leu Phe Ser Ala Gly Phe Val Leu Arg Trp Gly Asp Pro Pro 915 920 925 Phe His Thr Ala Gly Arg Leu Ile Tyr Cys Ile Asp Ile Ile Phe Trp 930 935 940 Phe Ser Arg Leu Leu Asp Phe Phe Ala Val Asn Gln His Ala Gly Pro 945 950 955 960 Tyr Val Thr Met Ile Ala Lys Met Thr Ala Asn Met Phe Tyr Ile Val 965 970 975 Ile Ile Met Ala Ile Val Leu Leu Ser Phe Gly Val Ala Arg Lys Ala 980 985 990 Ile Leu Ser Pro Lys Glu Pro Pro Ser Trp Ser Leu Ala Arg Asp Ile 995 1000 1005 Val Phe Glu Pro Tyr Trp Met Ile Tyr Gly Glu Val Tyr Ala Gly 1010 1015 1020 Glu Ile Asp Gly Arg Pro Leu Pro Ser Gly Ala Leu Ala Leu Lys 1025 1030 1035 Ser Pro Trp Trp Gly Cys Pro Phe Arg Cys Pro Leu Ser Ile Ser 1040 1045 1050 Lys Ala Leu Ile Leu Asp Leu Glu Ala Lys Cys Ser Thr Asp Thr 1055 1060 1065 Arg Leu Gln Phe Pro Leu Gly Gln Leu Glu Val Ser Phe Leu Ala 1070 1075 1080 Pro Ala Gln Ala Ala Lys Pro Ile Leu Ala Ser Glu His Ala Glu 1085 1090 1095 Arg His Phe Val Gly Asn Val Tyr Leu Asp Met Glu Ser Ile Ser 1100 1105 1110 Asn Asn Leu Trp Lys Tyr Asn Arg Tyr Arg Tyr Ile Met Thr Tyr 1115 1120 1125 His Glu Lys Pro Trp Leu Pro Pro Pro Leu Ile Leu Leu Ser His 1130 1135 1140 Val Gly Leu Leu Leu Arg Arg Leu Cys Cys His Arg Ala Pro His 1145 1150 1155 Asp Gln Glu Glu Gly Asp Val Gly Leu Lys Leu Tyr Leu Ser Lys 1160 1165 1170 Glu Asp Leu Lys Lys Leu His Asp Phe Glu Glu Gln Cys Val Glu 1175 1180 1185 Lys Tyr Phe His Glu Lys Met Glu Asp Val Asn Cys Ser Cys Glu 1190 1195 1200 Glu Arg Ile Arg Val Thr Ser Glu Ser Gly Val Gly Gly Cys Phe 1205 1210 1215 Thr Glu Val Gly Glu Asp Arg Ile Gly Glu Ala Glu Thr Ala Ile 1220 1225 1230 Thr Gly Gly Phe Trp Glu Lys Tyr Tyr Gly Ala Gly Lys Leu Gly 1235 1240 1245 Met Gly Thr Arg Val Thr Glu Met Tyr Phe Gln Leu Lys Glu Met 1250 1255 1260 Asn Glu Lys Val Ser Phe Ile Lys Asp Ser Leu Leu Ser Leu Asp 1265 1270 1275 Ser Gln Val Gly His Leu Gln Asp Leu Ser Ala Leu Thr Val Asp 1280 1285 1290 Thr Leu Lys Val Leu Ser Ala Val Asp Thr Leu Gln Glu Asp Glu 1295 1300 1305 Ala Leu Leu Ala Lys Arg Lys His Ser Thr Cys Lys Lys Leu Pro 1310 1315 1320 His Ser Trp Ser Asn Val Ile Cys Ala Glu Val Leu Gly Ser Met 1325 1330 1335 Glu Ile Ala Gly Glu Lys Lys Tyr Gln Tyr Tyr Ser Met Pro Ser 1340 1345 1350 Ser Leu Leu Arg Ser Leu Ala Gly Gly Arg His Pro Pro Arg Val 1355 1360 1365 Gln Arg Gly Ala Leu Leu Glu Ile Thr Asn Ser Lys Arg Glu Ala 1370 1375 1380 Thr Asn Val Arg Asn Asp Gln Glu Arg Gln Glu Thr Gln Ser Ser 1385 1390 1395 Ile Val Val Ser Gly Val Ser Pro Asn Arg Gln Ala His Ser Lys 1400 1405 1410 Tyr Gly Gln Phe Leu Leu Val Pro Ser Asn Leu Lys Arg Val Pro 1415 1420 1425 Phe Ser Ala Glu Thr Val Leu Pro Leu Ser Arg Pro Ser Val Pro 1430 1435 1440 Asp Val Leu Ala Thr Glu Gln Asp Ile Gln Thr Glu Val Leu Val 1445 1450 1455 His Leu Thr Gly Gln Thr Pro Val Val Ser Asp Trp Ala Ser Val 1460 1465 1470 Asp Glu Pro Lys Glu Lys His Glu Pro Ile Ala His Leu Leu Asp 1475 1480 1485 Gly Gln Asp Lys Ala Glu Gln Val Leu Pro Thr Leu Ser Cys Thr 1490 1495 1500 Pro Glu Pro Met Thr Met Ser Ser Pro Leu Ser Gln Ala Lys Ile 1505 1510 1515 Met Gln Thr Gly Gly Gly Tyr Val Asn Trp Ala Phe Ser Glu Gly 1520 1525 1530 Asp Glu Thr Gly Val Phe Ser Ile Lys Lys Lys Trp Gln Thr Cys 1535 1540 1545 Leu Pro Ser Thr Cys Asp Ser Asp Ser Ser Arg Ser Glu Gln His 1550 1555 1560 Gln Lys Gln Ala Gln Asp Ser Ser Leu Ser Asp Asn Ser Thr Arg 1565 1570 1575 Ser Ala Gln Ser Ser Glu Cys Ser Glu Val Gly Pro Trp Leu Gln 1580 1585 1590 Pro Asn Thr Ser Phe Trp Ile Asn Pro Leu Arg Arg Tyr Arg Pro 1595 1600 1605 Phe Ala Arg Ser His Ser Phe Arg Phe His Lys Glu Glu Lys Leu 1610 1615 1620 Met Lys Ile Cys Lys Ile Lys Asn Leu Ser Gly Ser Ser Glu Ile 1625 1630 1635 Gly Gln Gly Ala Trp Val Lys Ala Lys Met Leu Thr Lys Asp Arg 1640 1645 1650 Arg Leu Ser Lys Lys Lys Lys Asn Thr Gln Gly Leu Gln Val Pro 1655 1660 1665 Ile Ile Thr Val Asn Ala Cys Ser Gln Ser Asp Gln Leu Asn Pro 1670 1675 1680 Glu Pro Gly Glu Asn Ser Ile Ser Glu Glu Glu Tyr Ser Lys Asn 1685 1690 1695 Trp Phe Thr Val Ser Lys Phe Ser His Thr Gly Val Glu Pro Tyr 1700 1705 1710 Ile His Gln Lys Met Lys Thr Lys Glu Ile Gly Gln Cys Ala Ile 1715 1720 1725 Gln Ile Ser Asp Tyr Leu Lys Gln Ser Gln Glu Glu Asn Ala Asp 1730 1735 1740 His Ser Leu Leu Ile Lys Ile Gly Leu Arg Leu Ser Leu Glu Lys 1745 1750 1755 Glu Asp Glu Leu Cys Lys Val Asn Thr Gly Glu Glu Ile Thr Val 1760 1765 1770 Tyr Arg Leu Glu Glu Ser Ser Pro Leu Asn Leu Asp Lys Ser Met 1775 1780 1785 Ser Ser Trp Ser Gln Arg Gly Arg Ala Ala Met Ile Gln Val Leu 1790 1795 1800 Ser Arg Glu Glu Met Asp Gly Gly Leu Arg Lys Ala Met Arg Val 1805 1810 1815 Val Ser Thr Trp Ser Glu Asp Asp Ile Leu Lys Pro Gly Gln Val 1820 1825 1830 Phe Ile Val Lys Ser Phe Leu Pro Glu Val Val Arg Thr Trp His 1835 1840 1845 Lys Ile Phe Gln Glu Ser Thr Val Leu His Leu Cys Leu Arg Glu 1850 1855 1860 Ile Gln Gln Gln Arg Ala Ala Gln Lys Leu Ile Tyr Thr Phe Asn 1865 1870 1875 Gln Val Lys Pro Gln Thr Ile Pro Tyr Thr Pro Arg Phe Leu Glu 1880 1885 1890 Val Phe Leu Ile Tyr Cys His Ser Ala Asn Gln Trp Leu Thr Ile 1895 1900 1905 Glu Lys Tyr Met Thr Gly Glu Phe Arg Lys Tyr Asn Asn Asn Asn 1910 1915 1920 Gly Asp Glu Ile Thr Pro Thr Asn Thr Leu Glu Glu Leu Met Leu 1925 1930 1935 Ala Phe Ser His Trp Thr Tyr Glu Tyr Thr Arg Gly Glu Leu Leu 1940 1945 1950 Val Leu Asp Leu Gln Val Phe Gly Glu Ile Met His Ile Val Trp 1955 1960 1965 Val Val Glu Val Glu Thr Glu Ala His Gln Gln Gln Leu Gly Gly 1970 1975 1980 Gly Ala Leu Glu Leu Gln Cys Arg Ser Leu Thr Ser Gly Ser Thr 1985 1990 1995 Val Asn 2000

Claims (71)

1. An isolated polynucleotide being selected from the group consisting of:
a) a polynucleotide encoding a transient receptor potential channel polypeptide comprising an amino acid sequence selected form the group consisting of:
amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 2;
the amino acid sequence shown in SEQ ID NO: 2.
amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 11;
the amino acid sequence shown in SEQ ID NO: 11;
amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NO: 12 and;
the amino acid sequence shown in SEQ ID NO: 12,
b) a polynucleotide comprising the sequence of SEQ ID NOS: 1; 9 or 10;
c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a transient receptor potential channel polypeptide;
d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a transient receptor potential channel polypeptide; and
e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a transient receptor potential channel polypeptide.
2. An expression vector containing any polynucleotide of claim 1.
3. A host cell containing the expression vector of claim 2.
4. A substantially purified transient receptor potential channel polypeptide encoded by a polynucleotide of claim 1.
5. A method for producing a transient receptor potential channel polypeptide, wherein the method comprises the following steps:
a) culturing the host cell of claim 3 under conditions suitable for the expression of the transient receptor potential channel polypeptide; and
b) recovering the transient receptor potential channel polypeptide from the host cell culture.
6. A method for detection of a polynucleotide encoding a transient receptor potential channel polypeptide in a biological sample comprising the following steps:
a) hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and
b) detecting said hybridization complex.
7. The method of claim 6, wherein before hybridization, the nucleic acid material of the biological sample is amplified.
8. A method for the detection of a polynucleotide of claim 1 or a transient receptor potential channel polypeptide of claim 4 comprising the steps of:
contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the transient receptor potential channel polypeptide.
9. A diagnostic kit for conducting the method of any one of claims 6 to 8.
10. A method of screening for agents which decrease the activity of a transient receptor potential channel, comprising the steps of:
contacting a test compound with any transient receptor potential channel polypeptide encoded by any polynucleotide of claim1;
detecting binding of the test compound to the transient receptor potential channel polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a transient receptor potential channel.
11. A method of screening for agents which regulate the activity of a transient receptor potential channel, comprising the steps of:
contacting a test compound with a transient receptor potential channel polypeptide encoded by any polynucleotide of claim 1; and
detecting a transient receptor potential channel activity of the polypeptide, wherein a test compound which increases the transient receptor potential channel activity is identified as a potential therapeutic agent for increasing the activity of the transient receptor potential channel, and wherein a test compound which decreases the transient receptor potential channel activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the transient receptor potential channel.
12. A method of screening for agents which decrease the activity of a transient receptor potential channel, comprising the steps of:
contacting a test compound with any polynucleotide of claim 1 and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of transient receptor potential channel.
13. A method of reducing the activity of transient receptor potential channel, comprising the steps of:
contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 or any transient receptor potential channel polypeptide of claim 4, whereby the activity of transient receptor potential channel is reduced.
14. A reagent that modulates the activity of a transient receptor potential channel polypeptide or a polynucleotide wherein said reagent is identified by the method of any of the claim 10 to 12.
15. A pharmaceutical composition, comprising:
the expression vector of claim 2 or the reagent of claim 14 and a pharmaceutically acceptable carrier.
16. Use of the expression vector of claim 2 or the reagent of claim 14 in the preparation of a medicament for modulating the activity of a transient receptor potential channel in a disease.
17. Use of claim 16 wherein the disease is cancer, a cardivascular disorder or a CNS disorder.
18. A cDNA encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NOS:2, 11 or 12.
19. The cDNA of claim 18 which comprises SEQ ID NOS:1; 9 or 10.
20. The cDNA of claim 18 which consists of SEQ ID NOS:1; 9 or 10.
21. An expression vector comprising a polynucleotide which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NOS:2; 11 or 12.
22. The expression vector of claim 21 wherein the polynucleotide consists of SEQ ID NOS:1; 9 or 10.
23. A host cell comprising an expression vector which encodes a polypeptide comprising the amino acid sequence shown in SEQ ID NOS:2; 11 or 12.
24. The host cell of claim 23 wherein the polynucleotide consists of SEQ ID NOS:1; 9 or 10.
25. A purified polypeptide comprising the amino acid sequence shown in SEQ ID NOS:2; 11 or 12.
26. The purified polypeptide of claim 25 which consists of the amino acid sequence shown in SEQ ID NOS:2; 11 or 12.
27. A fusion protein comprising a polypeptide having the amino acid sequence shown in SEQ ID NOS:2; 11 or 12.
28. A method of producing a polypeptide comprising the amino acid sequence shown in SEQ ID NOS:2; 11 or 12 comprising the steps of:
culturing a host cell comprising an expression vector which encodes the polypeptide under conditions whereby the polypeptide is expressed; and
isolating the polypeptide.
29. The method of claim 28 wherein the expression vector comprises SEQ ID NOS:1; 9 or 10.
30. A method of detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID NOS:2; 11 or 12, comprising the steps of:
hybridizing a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NOS:1; 9 or 10 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and
detecting the hybridization complex.
31. The method of claim 30 further comprising the step of amplifying the nucleic acid material before the step of hybridizing.
32. A kit for detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID NOS:2; 11 or 12, comprising:
a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NOS:1; 9 or 10; and
instructions for the method of claim 30.
33. A method of detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NOS:2; 11 or 12 comprising the steps of:
contacting a biological sample with a reagent that specifically binds to the polypeptide to form a reagent-polypeptide complex; and
detecting the reagent-polypeptide complex.
34. The method of claim 33 wherein the reagent is an antibody.
35. A kit for detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NOS:2; 11 or 12 comprising:
an antibody which specifically binds to the polypeptide; and
instructions for the method of claim 33.
36. A method of screening for agents which can modulate the activity of a human transient receptor potential channel, comprising the steps of:
contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NOS:2; 11 or 12 and (2) the amino acid sequence shown in SEQ ID NOS:2; 11 or 12; and
detecting binding of the test compound to the polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential agent for regulating activity of the human transient receptor potential channel.
37. The method of claim 36 wherein the step of contacting is in a cell.
38. The method of claim 36 wherein the cell is in vitro.
39. The method of claim 36 wherein the step of contacting is in a cell-free system.
40. The method of claim 36 wherein the polypeptide comprises a detectable label.
41. The method of claim 36 wherein the test compound comprises a detectable label.
42. The method of claim 36 wherein the test compound displaces a labeled ligand which is bound to the polypeptide.
43. The method of claim 36 wherein the polypeptide is bound to a solid support.
44. The method of claim 36 wherein the test compound is bound to a solid support.
45. A method of screening for agents which modulate an activity of a human transient receptor potential channel, comprising the steps of:
contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 26% identical to the amino acid sequence shown in SEQ ID NOS:2; 11 or 12 and (2) the amino acid sequence shown in SEQ ID NOS:2; 11 or 12 and
detecting an activity of the polypeptide, wherein a test compound which increases the activity of the polypeptide is identified as a potential agent for increasing the activity of the human transient receptor potential channel, and wherein a test compound which decreases the activity of the polypeptide is identified as a potential agent for decreasing the activity of the human transient receptor potential channel.
46. The method of claim 45 wherein the step of contacting is in a cell.
47. The method of claim 45 wherein the cell is in vitro.
48. The method of claim 45 wherein the step of contacting is in a cell-free system.
49. A method of screening for agents which modulate an activity of a human transient receptor potential channel, comprising the steps of:
contacting a test compound with a product encoded by a polynucleotide which comprises the nucleotide sequence shown in SEQ ID NOS:1; 9 or 10; and
detecting binding of the test compound to the product, wherein a test compound which binds to the product is identified as a potential agent for regulating the activity of the human transient receptor potential channel.
50. The method of claim 49 wherein the product is a polypeptide.
51. The method of claim 49 wherein the product is RNA.
52. A method of reducing activity of a human transient receptor potential channel, comprising the step of:
contacting a cell with a reagent which specifically binds to a product encoded by a polynucleotide comprising the nucleotide sequence shown in SEQ ID NOS:1; 9 or 10, whereby the activity of a human transient receptor potential channel is reduced.
53. The method of claim 52 wherein the product is a polypeptide.
54. The method of claim 53 wherein the reagent is an antibody.
55. The method of claim 52 wherein the product is RNA.
56. The method of claim 55 wherein the reagent is an antisense oligonucleotide.
57. The method of claim 56 wherein the reagent is a ribozyme.
58. The method of claim 52 wherein the cell is in vitro.
59. The method of claim 52 wherein the cell is in vivo.
60. A pharmaceutical composition, comprising:
a reagent which specifically binds to a polypeptide comprising the amino acid sequence shown in SEQ ID NOS:2; 11 or 12; and
a pharmaceutically acceptable carrier.
61. The pharmaceutical composition of claim 60 wherein the reagent is an antibody.
62. A pharmaceutical composition, comprising:
a reagent which specifically binds to a product of a polynucleotide comprising the nucleotide sequence shown in SEQ ID NOS:1; 9 or 10; and
a pharmaceutically acceptable carrier.
63. The pharmaceutical composition of claim 62 wherein the reagent is a ribozyme.
64. The pharmaceutical composition of claim 62 wherein the reagent is an antisense oligonucleotide.
65. The pharmaceutical composition of claim 62 wherein the reagent is an antibody.
66. A pharmaceutical composition, comprising:
an expression vector encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NOS:2; 11 or 12; and
a pharmaceutically acceptable carrier.
67. The pharmaceutical composition of claim 66 wherein the expression vector comprises SEQ ID NOS:1; 9 or 10.
68. A method of treating a transient receptor potential channel dysfunction related disease, wherein the disease is selected from cancer, a cardiovascular disorder or a CNS disorder comprising the step of:
administering to a patient in need thereof a therapeutically effective dose of a reagent that modulates a function of a human transient receptor potential channel, whereby symptoms of the transient receptor potential channel disfunction related disease are ameliorated.
69. The method of claim 68 wherein the reagent is identified by the method of claim 36.
70. The method of claim 68 wherein the reagent is identified by the method of claim 45.
71. The method of claim 68 wherein the reagent is identified by the method of claim 49.
US10/467,163 2002-02-19 2002-02-19 Regulation of human transient receptor potential channel Abandoned US20040115675A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060217340A1 (en) * 2005-03-23 2006-09-28 University Of Vermont And State Agricultural College Methods and products for treating hypertension by modulation of TRPC3 channel activity
WO2023009615A3 (en) * 2021-07-28 2023-03-23 University Of Connecticut Methods and compositions for treating or preventing neurological injury or neurological disorders

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020177205A1 (en) * 2000-08-03 2002-11-28 Alexey Ryazanov Mammalian alpha-kinase proteins, nucleic acids and diagnostic and therapeutic uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020177205A1 (en) * 2000-08-03 2002-11-28 Alexey Ryazanov Mammalian alpha-kinase proteins, nucleic acids and diagnostic and therapeutic uses thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060217340A1 (en) * 2005-03-23 2006-09-28 University Of Vermont And State Agricultural College Methods and products for treating hypertension by modulation of TRPC3 channel activity
WO2023009615A3 (en) * 2021-07-28 2023-03-23 University Of Connecticut Methods and compositions for treating or preventing neurological injury or neurological disorders

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